九州大学 研究者情報
論文一覧
友清 淳(ともきよ あつし) データ更新日:2022.05.23

講師 /  九州大学病院 口腔機能修復科 口腔機能修復学講座


原著論文
1. 山下 梢, 友清 淳, 小野 太雅, 一法師 啓太, 濱野 さゆり, 杉井 英樹, 吉田 晋一郎, 糸山 知宏, 前田 英史, Iodine potassium iodideがWhite Mineral Trioxide Aggregateの表面性状および機械的強度に及ぼす影響について, 日本歯内療法学会雑誌, https://doi.org/10.20817/jeajournal.42.3_181, 42, 3, 181-187, 2021.10.
2. Ipposhi Keita; Tomokiyo Atsushi; Ono Taiga; Yamashita Kozue; Alhasan Muhammad Anas; Hasegawa Daigaku; Hamano Sayuri; Yoshida Shinichiro; Sugii Hideki; Itoyama Tomohiro; Ogawa Marina; Maeda Hidefumi, Secreted Frizzled-Related Protein 1 Promotes Odontoblastic Differentiation and Reparative Dentin Formation in Dental Pulp Cells, CELLS, 10.3390/cells10092491, 10, 9, 2021.09.
3. Tomokiyo Atsushi; Hasegawa Daigaku; Ono Taiga; Nagano Ryoko; Ipposhi Keita; Yamashita Kozue; Alhasan M. Anas; Maeda Hidefumi, Characterization of a clonal human periodontal ligament stem cell line exposed to methacrylate resin-, bioactive glass-, or silicon-based root canal sealers, ODONTOLOGY, 10.1007/s10266-021-00648-7, 2021.08.
4. Ono, Taiga; Tomokiyo, Atsushi; Ipposhi, Keita; Yamashita, Kozue; Alhasan, M. Anas; Miyazaki, Yudai; Kunitomi, Yoshihiro; Tsuchiya, Akira; Ishikawa, Kunio; Maeda, Hidefumi, Generation of biohybrid implants using a multipotent human periodontal ligament cell line and bioactive core materials, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.30336, 2021.02.
5. Tomohiro Itoyama, Shinichiro Yoshida, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Taiga Ono, Shoko Fujino, Hidefumi Maeda., Possible function of GDNF and Schwann cells in wound healing of periodontal tissue, Journal of periodontal research, 2020.12.
6. Hasegawa, Daigaku; Hasegawa, Kana; Kaneko, Hiroshi; Yoshida, Shinichiro; Mitarai, Hiromi; Arima, Mai; Tomokiyo, Atsushi; Hamano, Sayuri; Sugii, Hideki; Wada, Naohisa; Kiyoshima, Tamotsu; Maeda, Hidefumi, MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells, STEM CELLS INTERNATIONAL, 10.1155/2020/9672673, 2020, 2020.07.
7. Tomohiro Itoyama , Shinichiro Yoshida , Atsushi Tomokiyo , Daigaku Hasegawa , Sayuri Hamano , Hideki Sugii , Taiga Ono , Shoko Fujino , Hidefumi Maeda, Possible function of GDNF and Schwann cells in wound healing of periodontal tissue, JOURNAL OF PERIODONTAL RESEARCH, 10.1111/jre.12774, 2020.06, Objective: The purpose of this study was to evaluate the function of Schwann cells in wound healing of periodontal tissue.

Background: In our previous study, glial cell line-derived neurotrophic factor (GDNF) promoted the migration of human periodontal ligament (PDL) cells and that GDNF expression increased in wounded periodontal tissue. GDNF reportedly induces the migration of Schwann cell precursors. Schwann cells play a crucial role in the regeneration of peripheral tissues, including bone tissue. However, the role of Schwann cells on periodontal tissue regeneration remains unclear.

Methods: A transwell assay and a WST-1 (water-soluble tetrazolium compound-1) proliferation assay were used to determine whether GDNF promotes the migration and proliferation of Schwann cells, respectively. Quantitative RT-PCR and Alizarin Red S staining were performed to examine the effect of these cells on the differentiation of human preosteoblast (Saos2 cells) using conditioned medium from YST-1 (YST-1-CM). Western blotting analysis was performed to determine whether YST-1-CM activates ERK signaling pathway in Saos2 cells. The expression of Schwann cell markers, S100 calcium-binding protein B (S100-B) and growth associated protein 43 (GAP-43), was determined in normal and wounded periodontal tissue by immunofluorescent staining.

Results: Glial cell line-derived neurotrophic factor promoted the migration of YST-1 cells but did not affect the proliferation of YST-1 cells. Saos2 cells cultured with YST-1-CM increased the expression of osteoblastic markers and mineralization. YST-1-CM also induced phosphorylation of ERK1/2 in Saos2 cells. The number of S100-B-immunoreactive cells which also expressed GAP-43 was increased in rat wounded periodontal tissue during healing process.

Conclusion: The accumulation of Schwann cells in wounded periodontal tissue suggests that they play a significant role in wound healing of this tissue, especially alveolar bone tissue..
8. Aoi Nozu, Sayuri Hamano, Atsushi Tomokiyo, Daigaku Hasegawa, Hideki Sugii, Shinichiro Yoshida, Hiromi Mitarai, Shuntaro Taniguchi, Naohisa Wada, Hidefumi Maeda, Senescence and odontoblastic differentiation of dental pulp cells, Journal of cellular physiology, 10.1002/jcp.26905, 234, 1, 849-859, 2018.01, [URL], Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α..
9. Sayuri Hamano, Atsushi Tomokiyo, Daigaku Hasegawa, Shinichiro Yoshida, Hideki Sugii, Hiromi Mitarai, Shoko Fujino, Naohisa Wada, Hidefumi Maeda, Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells, Stem cells and development, 10.1089/scd.2017.0077, 27, 2, 100-111, 2018.01, [URL], The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration..
10. Mai Arima, Daigaku Hasegawa, Shinichiro Yoshida, Hiromi Mitarai, Atsushi Tomokiyo, Sayuri Hamano, Hideki Sugii, Naohisa Wada, Hidefumi Maeda, R-spondin 2 promotes osteoblastic differentiation of immature human periodontal ligament cells through the Wnt/β-catenin signaling pathway, Journal of Periodontal Research, 10.1111/jre.12611, 54, 2, 143-153, 2019.04, [URL], Objective: In this study, we measured the expression of R-spondin 2 (RSPO2) in periodontal ligament (PDL) tissue and cells. Further, we examined the effects of RSPO2 on osteoblastic differentiation of immature human PDL cells (HPDLCs). Background: R-spondin (RSPO) family proteins are secreted glycoproteins that play important roles in embryonic development and tissue homeostasis through activation of the Wnt/β-catenin signaling pathway. RSPO2, a member of the RSPO family, has been reported to enhance osteogenesis in mice. However, little is known regarding the roles of RSPO2 in PDL tissues. Methods: Expression of RSPO2 in rat PDL tissue and primary HPDLCs was examined by immunohistochemical and immunofluorescence staining, as well as by semiquantitative RT-PCR. The effects of stretch loading on the expression of RSPO2 and Dickkopf-related protein 1 (DKK1) were assessed by quantitative RT-PCR. Expression of receptors for RSPOs, such as Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4, 5, and 6 in immature human PDL cells (cell line 2-14, or 2-14 cells), was investigated by semiquantitative RT-PCR. Mineralized nodule formation in 2-14 cells treated with RSPO2 under osteoblastic inductive condition was examined by Alizarin Red S and von Kossa stainings. Nuclear translocation of β-catenin and expression of active β-catenin in 2-14 cells treated with RSPO2 were assessed by immunofluorescence staining and Western blotting analysis, respectively. In addition, the effect of Dickkopf-related protein 1 (DKK1), an inhibitor of Wnt/β-catenin signaling, was also examined. Results: Rat PDL tissue and HPDLCs expressed RSPO2, and HPDLCs also expressed RSPO2, while little was found in 2-14 cells. Expression of RSPO2 as well as DKK1 in HPDLCs was significantly upregulated by exposure to stretch loading. LGR4 was predominantly expressed in 2-14 cells, which expressed low levels of LGR5 and LGR6. RSPO2 enhanced the Alizarin Red S and von Kossa-positive reactions in 2-14 cells. In addition, DKK1 suppressed nuclear translocation of β-catenin, activation of β-catenin, and increases of Alizarin Red S and von Kossa-positive reactions in 2-14 cells, all of which were induced by RSPO2 treatment. Conclusion: RSPO2, which is expressed in PDL tissue and cells, might play an important role in regulating the osteoblastic differentiation of immature human PDL cells through the Wnt/β-catenin signaling pathway..
11. Sayuri Hamano, Atsushi Tomokiyo, Daigaku Hasegawa, Asuka Yuda, Hideki Sugii, Shinichiro Yoshida, Hiromi Mitarai, Naohisa Wada, Hidefumi Maeda, Functions of beta2-adrenergic receptor in human periodontal ligament cells, Journal of Cellular Biochemistry, 10.1002/jcb.29706, 2020.01, [URL], Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue..
12. Tomokiyo, Atsushi; Wada, Naohisa; Maeda, Hidefumi, Periodontal Ligament Stem Cells: Regenerative Potency in Periodontium, STEM CELLS AND DEVELOPMENT, 10.1089/scd.2019.0031, 28, 15, 974-985, 2019.08.
13. Fujino, Shoko; Hamano, Sayuri; Tomokiyo, Atsushi; Itoyama, Tomohiro; Hasegawa, Daigaku; Sugii, Hideki; Yoshida, Shinichiro; Washio, Ayako; Nozu, Aoi; Ono, Taiga; Wada, Naohisa; Kitamura, Chiaki; Maeda, Hidefumi, Expression and function of dopamine in odontoblasts, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.29314, 2019.10.
14. Atsushi Tomokiyo, Shinichiro Yoshida, Sayuri Hamano, Daigaku Hasegawa, Hideki Sugii, Hidefumi Maeda, Detection, characterization, and clinical application of mesenchymal stem cells in periodontal ligament tissue, Stem Cells International, 10.1155/2018/5450768, 2018, 2018.01, [URL], Mesenchymal stem cells (MSCs) are a kind of somatic stem cells that exert a potential to differentiate into multiple cell types and undergo robust clonal self-renewal; therefore, they are considered as a highly promising stem cell population for tissue engineering. MSCs are identified in various adult organs including dental tissues. Periodontal ligament (PDL) is a highly specialized connective tissue that surrounds the tooth root. PDL also contains MSC population, and many researchers have isolated them and performed their detailed characterization. Here, we review the current understanding of the features and functions of MSC population in PDL tissues and discuss their possibility for the application of PDL regeneration..
15. Arima M, Hasegawa D, Yoshida S, Mitarai H, Tomokiyo A, Hamano S, Sugii H, Wada N, Maeda H., R-spondin 2 promotes osteoblastic differentiation of immature human periodontal ligament cells through the Wnt/β-catenin signaling pathway., J Periodontal Res., 2018 Oct 4. doi: 10.1111/jre.12611., 2018.10.
16. Nozu A, Hamano S, Tomokiyo A, Hasegawa D, Sugii H, Yoshida S, Mitarai H, Taniguchi S, Wada N, Maeda H., Senescence and odontoblastic differentiation of dental pulp cells., J Cell Physiol., Jan;234(1):849-859, 2018.01.
17. Daigaku Hasegawa, Naohisa Wada, Shinichiro Yoshida, Hiromi Mitarai, Mai Arima, Atsushi Tomokiyo, Sayuri Hamano, Hideki Sugii, Hidefumi Maeda, Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell-like cells via Ror2/JNK signaling, Journal of Cellular Physiology, 10.1002/jcp.26086, 233, 2, 1752-1762, 2018.02, [URL], Wnt5a, a non-canonical Wnt protein, is known to play important roles in several cell functions. However, little is known about the effects of Wnt5a on osteoblastic differentiation of periodontal ligament (PDL) cells. Here, we examined the effects of Wnt5a on osteoblastic differentiation and associated intracellular signaling in human PDL stem/progenitor cells (HPDLSCs). We found that Wnt5a suppressed expression of bone-related genes (ALP, BSP, and Osterix) and alizarin red-positive mineralized nodule formation in HPDLSCs under osteogenic conditions. Immunohistochemical analysis revealed that a Wnt5a-related receptor, receptor tyrosine kinase-like orphan receptor 2 (Ror2), was expressed in rat PDL tissue. Interestingly, knockdown of Ror2 by siRNA inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Moreover, Western blotting analysis showed that phosphorylation of the intracellular signaling molecule, c-Jun N-terminal kinase (JNK) was upregulated in HPDLSCs cultured in osteoblast induction medium with Wnt5a, but knockdown of Ror2 by siRNA downregulated the phosphorylation of JNK. We also examined the effects of JNK inhibition on Wnt5a-induced suppression of osteoblastic differentiation of HPDLSCs. The JNK inhibitor, SP600125 inhibited the Wnt5a-induced downregulation of bone-related gene expression in HPDLSCs. Additionally, SP600125 inhibited the Wnt5a-induced suppression of the alizarin red-positive reaction in HPDLSCs. These results suggest that Wnt5a suppressed osteoblastic differentiation of HPDLSCs through Ror2/JNK signaling. Non-canonical Wnt signaling, including Wnt5a/Ror2/JNK signaling, may function as a negative regulator of mineralization, preventing the development of non-physiological mineralization in PDL tissue..
18. Sayuri Hamano, Atsushi Tomokiyo, Daigaku Hasegawa, Shinichiro Yoshida, Hideki Sugii, Hidefumi Maeda, Hiromi Mitarai, Shoko Fujino, Naohisa Wada, Hidefumi Maeda, Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells., Stem Cells and Development, 100-111, 2018.01.
19. H. Mitarai, Naohisa Wada, Daigaku Hasegawa, Shinichiro Yoshida, M. Sonoda, Atsushi Tomokiyo, Sayuri Hamano, S. Serita, H. Mizumachi, Hidefumi Maeda, Transgelin mediates transforming growth factor-β1-induced proliferation of human periodontal ligament cells, Journal of Periodontal Research, 10.1111/jre.12466, 52, 6, 984-993, 2017.12, [URL], Background and Objective: Human periodontal ligament cells (HPDLCs) express transforming growth factor-β1 (TGF-β1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells. Material and Methods: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription–polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-β1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-β1 were assessed by WST-1 assay. Results: In microarray and quantitative reverse transcription–polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-β1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-β1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-β1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA. Conclusion: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-β1 stimulation..
20. 友清淳、濱野さゆり、長谷川大学、杉井英樹、吉田晋一郎、御手洗裕美、有馬麻衣、野津葵、和田尚久、前田英史, 歯内治療ならびに修復処置関連溶液によって生じるWhite Mineral Trioxide Aggregateの色調変化に関する比較分析., 日本歯内療法学会誌, 60, 4, 200-210, 2017.08.
21. Hiroyuki Mizumachi, Shinichiro Yoshida, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Asuka Yuda, Hideki Sugii, Suguru Serita, Hiromi Mitarai, Katsuaki Koori, Naohisa Wada, Hidefumi Maeda, Calcium-sensing receptor-ERK signaling promotes odontoblastic differentiation of human dental pulp cells, Bone, 10.1016/j.bone.2017.05.012, 101, 191-201, 2017.08, [URL], Activation of the G protein-coupled calcium-sensing receptor (CaSR) has crucial roles in skeletal development and bone turnover. Our recent study has identified a role for activated CaSR in the osteogenic differentiation of human periodontal ligament stem cells. Furthermore, odontoblasts residing inside the tooth pulp chamber play a central role in dentin formation. However, it remains unclear how CaSR activation affects the odontoblastic differentiation of human dental pulp cells (HDPCs). We have investigated the odontoblastic differentiation of HDPCs exposed to elevated levels of extracellular calcium (Ca) and strontium (Sr), and the contribution of CaSR and the L-type voltage-dependent calcium channel (L-VDCC) to this process. Immunochemical staining of rat dental pulp tissue demonstrated that CaSR was expressed at high levels in the odontoblastic layer, moderate levels in the sublayer, and low levels in the central pulp tissue. Although normal HDPCs expressed low levels of CaSR, stimulation with Ca or Sr promoted both CaSR expression and odontoblastic differentiation of HDPCs along with increased expression of odontoblastic makers. These effects were inhibited by treatment with a CaSR antagonist, whereas treatment with an L-VDCC inhibitor had no effect. Additionally, knockdown of CaSR with siRNA suppressed odontoblastic differentiation of Ca- and Sr-treated HDPCs. ERK1/2 phosphorylation was observed in Ca- and Sr-treated HDPCs, whereas CaSR antagonist treatment or CaSR knockdown blocked ERK1/2 phosphorylation. Furthermore, inhibition of ERK1/2 suppressed mineralization of Ca- and Sr-treated HDPCs. These results suggest that elevated concentrations of extracellular Ca and Sr induce odontoblastic differentiation of HDPCs through CaSR activation and the ERK1/2 phosphorylation..
22. Daigaku Hasegawa, Naohisa Wada, Shinichiro Yoshida, Hiromi Mitarai, Mai Arima, Atsushi Tomokiyo, Sayuri Hamano, Hideki Sugii, Hidefumi Maeda, Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell-like cells via Ror2/JNK signaling., Journal of Cellular Physiology, 2017.07.
23. Suguru Serita, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Shinichiro Yoshida, Hiroyuki Mizumachi, Hiromi Mitarai, Monnouchi Satoshi, Naohisa Wada, Hidefumi Maeda, Transforming growth factor-β-induced gene product-h3 inhibits odontoblastic differentiation of dental pulp cells., Arch Oral Biol., 10.1016/j.archoralbio.2017.02.018, 78, 135-143, 2017.05, OBJECTIVE:
The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs).
DESIGN:
A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR.
RESULTS:
Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs.
CONCLUSIONS:
The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process..
24. Shinichiro Yoshida, Naohide Yamamoto, Naohisa Wada, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hiromi Mitarai, Satoshi Monnouchi, Asuka Yuda, Hidefumi Maeda, GDNF From Human Periodontal Ligament Cells Treated With Pro-Inflammatory Cytokines Promotes Neurocytic Differentiation of PC12 Cells, Journal of Cellular Biochemistry, 10.1002/jcb.25662, 118, 4, 699-708, 2017.04, [URL], Glial cell line-derived neurotrophic factor (GDNF) is known to mediate multiple biological activities such as promotion of cell motility and proliferation, and morphogenesis. However, little is known about its effects on periodontal ligament (PDL) cells. Recently, we reported that GDNF expression is increased in wounded rat PDL tissue and human PDL cells (HPDLCs) treated with pro-inflammatory cytokines. Here, we investigated the associated expression of GDNF and the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in wounded PDL tissue, and whether HPDLCs secrete GDNF which affects neurocytic differentiation. Rat PDL cells near the wounded area showed intense immunoreactions against an anti-GDNF antibody, where immunoreactivity was also increased against an anti-IL-1β antibody. Compared with untreated cells, HPDLCs treated with IL-1β or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1β-treated HPDLCs (IL-1β-CM) increased neurite outgrowth of PC12 rat adrenal pheochromocytoma cells. The expression levels of two neural regeneration-associated genes, growth-associated protein-43 (Gap-43), and small proline-rich repeat protein 1A (Sprr1A), were also upregulated in IL-1β-CM-treated PC12 cells. These stimulatory effects of IL-1β-CM were significantly inhibited by a neutralizing antibody against GDNF. In addition, U0126, a MEK inhibitor, inhibited GDNF-induced neurite outgrowth of PC12 cells. These findings suggest that an increase of GDNF in wounded PDL tissue might play an important role in neural regeneration probably via the MEK/ERK signaling pathway. J. Cell. Biochem. 118: 699–708, 2017..
25. Atsushi Tomokiyo, Kim Hynes, Jia Ng, Danijela Menicanin, Esther Camp, Agnes Arthur, Stan Gronthos, Peter Mark Bartold, Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells, Journal of Cellular Physiology, 10.1002/jcp.25437, 232, 2, 402-416, 2017.02, [URL], Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402–416, 2017..
26. S. Monnouchi, Hidefumi Maeda, A. Yuda, S. Serita, Naohisa Wada, Atsushi Tomokiyo, A. Akamine, Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells, Journal of Periodontal Research, 10.1111/jre.12355, 51, 6, 779-788, 2016.12, [URL], Background and Objective: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs. Material and Methods: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription–polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), which is an AhR antagonist. Results: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs. Conclusions: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking..
27. Shinichiro Yoshida, Naohide Yamamoto, Naohisa Wada, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hiromi Mitarai, Satoshi Monouchi, Asuka Yuda, Hidefumi Maeda, GDNF from Human Periodontal Ligament Cells Treated with Proinflammatory Cytokines Promotes Neurocytic Differentiation of PC12 Cells., Journal of Cellular Biochemistry, 2016.07.
28. Shinichiro Yoshida, Naohisa Wada, Daigaku Hasegawa, Hirofumi Miyaji, Hiromi Mitarai, Atsushi Tomokiyo, Sayuri Hamano, Hidefumi Maeda, Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells., J Dent Res, 10.1177/0022034516653085, 2016.06.
29. Atsushi Tomokiyo, Kim Hynes, Jia Ng, Danijela Menicanin, Agnes Arthur, Esther Camp, Stan Gronthos, P. Mark Bartold, Generation of neural crest-like cells from human periodontal ligament cell-derived induced pluripotent stem cells., Journal of Cellular Physiology, 10.1002, 2016.05, Neural crest cells (NCC) are a transient multipotent stem cell population that migrates into its destination in the vertebrate embryo and gives rise to many different types of cells. NCC hold great promise for stem cell-based tissue engineering, however the inability to easily obtain large numbers of neural crest cells is a major factor limiting their use in studies of development and regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC because of their self-renewal capacity and pluripotency. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with neural crest characteristics. Using magnetic-activated cell sorting, we isolated PDL iPSC- and FF iPSC-derived NCLC which highly expressed HNK1. The isolated NCLC also expressed the neural crest markers SLUG and SOX9. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers; CD73, CD90, CD105, CD146, and CD166, whilst very few cells expressed the pluripotency markers; TRA160, alkaline phosphatase, or the hematopoietic markers CD14, CD34, and CD45. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC..
30. Toyoda, Kyosuke, Fukuda, Takao, Sanui, Terukazu, Tanaka, Urara, Yamamichi, Kensuke, Atomura, Ryo, Maeda, Hidefumi, Atsushi Tomokiyo, Taketomi, Takaharu, Uchiumi, Takeshi, Nishimura, Fusanori, Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.25087, 231, 2, 414-427, 2016.02, Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therap.
31. Satoshi Monnouchi, Hidefumi Maeda, Asuka Yuda, Suguru Serita, Naohisa Wada, Atsushi Tomokiyo, Akifumi Akamine, Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells., J Periodontal Res, 2016.01.
32. Atsushi Tomokiyo, Naohisa Wada, Hidefumi Maeda, Contribution of Stem Cells to Dental Tissue Regeneration: Isolation, Function, and Application., Frontiers in Stem Cell and Regenerative Medicine Research, 2, 3-38, 2016.
33. Atsushi Tomokiyo, Naohisa Wada, Sayuri Hamano, Daigaku Hasegawa, Hideki Sugii, Shinichiro Yoshida, Hidefumi Maeda, Periodontal Ligament Stem Cells in Regenerative Dentistry for Periodontal Tissues., Journal of Stem Cell Research & Therapy, 1, 3, 2016.
34. Hasegawa, Daigaku, Wada, Naohisa, Maeda, Hidefumi, Yoshida, Shinichiro, Mitarai, Hiromi, Atsushi Tomokiyo, Monnouchi, Satoshi, Hamano, Sayuri, Yuda, Asuka, Akamine, Akifumi, Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGF1-Mediated Upregulation of Periostin Expression, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.24950, 230, 11, 2647-2660, 2015.11, Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this.
35. Monnouchi Satoshi, Hidefumi Maeda, Asuka Yuda, Sayuri Hamano, Naohisa Wada, Atsushi Tomokiyo, Koori Katsuaki, Hideki Sugii, Shun Serita, Akifumi Akamine, Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells, JOURNAL OF PERIODONTAL RESEARCH, 10.1111/jre.12200, 50, 2, 231-239, 2015.04.
36. Daigaku Hasegawa, Naohisa Wada, Hidefumi Maeda, Shinichiro Yosihida, Hiromi Mitarai, Atsushi Tomokiyo, Monnouchi Satoshi, Sayuri Hamano, Asuka Yuda, Akifumi Akamine, Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells through TGFβ1-mediated Upregulation of Periostin Expression., J Cell Physiol, 2015.02.
37. Asuka Yuda, Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohide Yamamoto, Naohisa Wada, Katsuaki Koori, Atsushi Tomokiyo, Sayuri Hamano, Daigaku Hasegawa, Akifumi Akamine, Effect of CTGF/CCN2 on osteo/ cementoblastic and fibroblastic differentiation of a human periodontal ligament stem/progenitor cell line, Journal of Cellular Physiology, 10.1002/jcp.24693, 230, 1, 150-159, 2015, [URL], Appropriate mechanical loading during occlusion and mastication play an important role in maintaining the homeostasis of periodontal ligament (PDL) tissue. Connective tissue growth factor (CTGF/CCN2), a matricellular protein, is known to upregulate extracellular matrix production, including collagen in PDL tissue. However, the underlying mechanisms of CTGF/CCN2 in regulation of PDL tissue integrity remain unclear. In this study, we investigated the effect of CTGF/CCN2 on osteo/cementoblastic and fibroblastic differentiation of human PDL stem cells using the cell line 1-11. CTGF/CCN2 expression in rat PDL tissue and human PDL cells (HPDLCs) was confirmed immunohisto/cytochemically. Mechanical loading was found to increase gene expression and secretion of CTGF/CCN2 in HPDLCs. CTGF/CCN2 upregulated the proliferation and migration of 1-11 cells. Furthermore, increased bone/cementum-related gene expression in this cell line led to mineralization. In addition, combined treatment of 1-11 cells with CTGF/CCN2 and transforming growth factor-β1 (TGF-β1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-β1 treatment alone. Thus, these data suggest the underlying biphasic effects of CTGF/CCN2 in 1-11 cells, inducible osteo/cementoblastic, and fibroblastic differentiation dependent on the environmental condition. CTGF/CCN2 may contribute to preservation of the structural integrity of PDL tissue, implying its potential use as a therapeutic agent for PDL regeneration..
38. Hidefumi Maeda, Atsushi Tomokiyo, Naohisa Wada, Katsuaki Koori, Giichiro Kawachi, Akifumi Akamine, Regeneration of the periodontium for preservation of the damaged tooth, Histology and Histopathology, 29, 10, 1249-1262, 2014.10, The population of the world grows every year, and life expectancy tends to increase. Thus, long-term preservation of teeth in aged individuals is an urgent issue. The main causes of tooth loss are well known to be periodontitis, caries, fractures, and orthodontic conditions. Although implant placement is a widely accepted treatment for tooth loss, most patients desire to preserve their own teeth. Many clinicians and researchers are therefore challenged to treat and preserve teeth that are irreversibly affected by deep caries, periodontitis, fractures, and trauma. Tissue engineering techniques are beneficial in addressing this issue; stem cells, signal molecules, and scaffolds are the main elements of such techniques. In this review, we describe these three elements with respect to their validation for regeneration of the periodontium and focus particularly on the potency of diverse scaffolds. In addition, we provide a short overview of the ongoing studies of 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl-borane resin including calcium chloride or hydroxyapatite for periodontium regeneration..
39. Yoko Teramatsu, Hidefumi Maeda, Hideki Sugii, Atsushi Tomokiyo, Sayuri Hamano, Naohisa Wada, Asuka Yuda, Naohide Yamamoto, Katsuaki Koori, Akifumi Akamine, Expression and effects of epidermal growth factor on human periodontal ligament cells, Cell and Tissue Research, 10.1007/s00441-014-1877-x, 357, 3, 633-643, 2014.09, [URL], Repair of damaged periodontal ligament (PDL) tissue is an essential challenge in tooth preservation. Various researchers have attempted to develop efficient therapies for healing and regenerating PDL tissue based on tissue engineering methods focused on targeting signaling molecules in PDL stem cells and other mesenchymal stem cells. In this context, we investigated the expression of epidermal growth factor (EGF) in normal and surgically wounded PDL tissues and its effect on chemotaxis and expression of osteoinductive and angiogenic factors in human PDL cells (HPDLCs). EGF as well as EGF receptor (EGFR) expression was observed in HPDLCs and entire PDL tissue. In a PDL tissue-injured model of rat, EGF and IL-1β were found to be upregulated in a perilesional pattern. Interleukin-1β induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-α (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) expression. Transwell assays demonstrated the chemotactic activity of EGF on HPDLCs. In addition, EGF treatment significantly induced secretion of bone morphogenetic protein 2 and vascular endothelial growth factor, and gene expression of interleukin-8 (IL-8), and early growth response-1 and -2 (EGR-1/2). Human umbilical vein endothelial cells developed well-formed tube networks when cultured with the supernatant of EGF-treated HPDLCs. These results indicated that EGF upregulated under inflammatory conditions plays roles in the repair of wounded PDL tissue, suggesting its function as a prospective agent to allow the healing and regeneration of this tissue..
40. Naohisa Wada, Hidefumi Maeda, Daigaku Hasegawa, Stan Gronthos, Peter Mark Bartold, Danijela Menicanin, Shinsuke Fujii, Shinichiro Yoshida, Atsushi Tomokiyo, Satoshi Monnouchi, Akifumi Akamine, Semaphorin 3A induces mesenchymal-stem-like properties in human periodontal ligament cells, Stem Cells and Development, 10.1089/scd.2013.0405, 23, 18, 2225-2236, 2014.09, [URL], Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells..
41. Katsuaki Koori, Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, Giichiro Kawachi, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Naohisa Wada, Akifumi Akamine, The roles of calcium-sensing receptor and calcium channel in osteogenic differentiation of undifferentiated periodontal ligament cells, Cell and Tissue Research, 10.1007/s00441-014-1918-5, 357, 3, 707-718, 2014.09, [URL], Elevated extracellular calcium has been shown to promote the differentiation of osteoblasts. However, the way that calcium affects the osteogenic differentiation of human periodontal ligament stem/progenitor cells (PDLSCs) remains unclear. Our aim has been to investigate the proliferation and osteogenic differentiation of a calcium-exposed human PDLSC line (cell line 1-17) that we have recently established and to elucidate the roles of the calcium-sensing receptor (CaSR) and L-type voltage-dependent calcium channel (L-VDCC) in this process. Proliferation activity was investigated by WST-1 assay, and gene and protein expression was examined by quantitative reverse transcriptase plus the polymerase chain reaction and immunostaining, respectively. Calcification assay was performed by von Kossa and Alizarin red staining. Treatment with 5 mM CaCl2 significantly induced proliferation, bone-related gene expression, and calcification in cell line 1-17. During culture with 5 mM CaCl2, this cell line up-regulated the gene expression of CaSR, which was reduced after 7 days. Simultaneous treatment with NPS2143, a CaSR inhibitor, and calcium significantly further increased bone-related gene expression and calcification as compared with CaCl2 exposure alone. The L-VDCC inhibitor, nifedipine, significantly suppressed osteogenic differentiation of cell line 1-17 treated with 5 mM CaCl2 and promoted the expression of CaSR, as compared with calcium treatment alone. Thus, elevated extracellular calcium promotes the proliferation and osteogenic differentiation of a PDLSC line. Antagonizing CaSR further enhances the effect of calcium on osteogenic differentiation, with CaSR expression being regulated by L-VDCC under extracellular calcium. Extracellular calcium might therefore modulate the osteogenic differentiation of PDLSCs through reciprocal adjustments of CaSR and L-VDCC..
42. Hideki Sugii, Hidefumi Maeda, Atsushi Tomokiyo, Naohide Yamamoto, Naohisa Wada, Katsuaki Koori, Daigaku Hasegawa, Sayuri Hamano, Asuka Yuda, Satoshi Monnouchi, Akifumi Akamine, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone, 10.1016/j.bone.2014.05.021, 66, 62-71, 2014.01, [URL], Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily and a dimer of inhibinβa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1β expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1β or TNF-α also up-regulated the expression of the gene encoding inhibinβa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction..
43. Hidefumi Maeda, Naohisa Wada, Atsushi Tomokiyo, Satoshi Monnouchi, Akifumi Akamine, Prospective potency of TGF-β1 on maintenance and regeneration of periodontal tissue, International Review of Cell and Molecular Biology, 10.1016/B978-0-12-407696-9.00006-3, 304, 283-367, 2013.07, [URL], Periodontal ligament (PDL) tissue, central in the periodontium, plays crucial roles in sustaining tooth in the bone socket. Irreparable damages of this tissue provoke tooth loss, causing a decreased quality of life. The question arises as to how PDL tissue is maintained or how the lost PDL tissue can be regenerated. Stem cells included in PDL tissue (PDLSCs) are widely accepted to have the potential to maintain or regenerate the periodontium, but PDLSCs are very few in number. In recent studies, undifferentiated clonal human PDL cell lines were developed to elucidate the applicable potentials of PDLSCs for the periodontal regenerative medicine based on cell-based tissue engineering. In addition, it has been suggested that transforming growth factor-beta 1 is an eligible factor for the maintenance and regeneration of PDL tissue..
44. Kiyomi Kono, Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, Naohide Yamamoto, Naohisa Wada, Satoshi Monnouchi, Yoko Teramatsu, Sayuri Hamano, Katsuaki Koori, Akifumi Akamine, Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines, Cell and Tissue Research, 10.1007/s00441-012-1543-0, 352, 2, 249-263, 2013.05, [URL], Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium..
45. Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, Naohisa Wada, Akifumi Akamine, Periodontal tissue engineering
Defining the triad, International Journal of Oral and Maxillofacial Implants, 10.11607/jomi.te26, 28, 6, e461-e471, 2013, [URL], The idea that somatic stem cells are localized in periodontal ligament (PDL) tissues as PDL stem cells (PDLSCs) responsible for construction and reconstruction of the periodontium has been widely accepted. Many dental scientists have attempted to clarify the identity of these PDLSCs but the number of PDLSCs localized in PDL tissues is too small to be routinely and conveniently analyzed. Therefore, researchers have been attempting to develop undifferentiated PDL cell lines by transducing them with genes that are suitable for immortalization. The present authors were the first to succeed in establishing two clonal human PDL stem/progenitor cell lines that possessed multipotency derived from PDL tissues and that expressed PDL-related molecules as well as neural crest-and embryonic stem-related markers. The differentiation stages of these cell lines appeared to vary based on their potential to differentiate into other lineage cells, their response to tissue regeneration-related cytokines, and their behavior when transplanted into immunodeficient rats. This review describes the phenotypes of these cell lines compared with reported PDLSCs or other MSCs and discusses contemporary circumstances related to PDL regenerative medicine. Differential analyses between these two clones will reveal the mechanism of differentiation of PDLSCs as well as their phenotypes. The results will also allow for the acquisition of a mass population of PDLSCs or other stem cells directed toward PDL-lineage cells and to develop an unmet treatment needed for construction and reconstruction of PDL tissues based on tissue engineering techniques..
46. Atsushi Tomokiyo, Hidefumi Maeda, Fujii Shinsuke, Monnouchi Satoshi, Naohisa Wada, Kono Kiyomi, Koori Katsuaki, naohide yamamoto, Yoko Teramatsu, Akifumi Akamine, Alternation of extracellular matrix remodeling and apoptosis by activation of the aryl hydrocarbon receptor pathway in human periodontal ligament cells, JOURNAL OF CELLULAR BIOCHEMISTRY, 10.1002/jcb.24186, 113, 10, 3093-3103, 2012.10.
47. naohide yamamoto, Hidefumi Maeda, Atsushi Tomokiyo, Fujii Shinsuke, Naohisa Wada, Monnouchi Satoshi, Kono Kiyomi, Koori Katsuaki, Yoko Teramatsu, Akifumi Akamine, Expression and effects of glial cell line-derived neurotrophic factor on periodontal ligament cells, JOURNAL OF CLINICAL PERIODONTOLOGY, 10.1111/j.1600-051X.2012.01881.x, 39, 6, 556-564, 2012.06.
48. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohisa Wada, Kiyomi Kono, Naohide Yamamoto, Katsuaki Koori, Yoko Teramatsu, Akifumi Akamine, A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival, Journal of Cellular Physiology, 10.1002/jcp.22933, 227, 5, 2040-2050, 2012.05, [URL], Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration..
49. Hidefumi Maeda, Atsushi Tomokiyo, Naohisa Wada, Akifumi Akamine, Induction of BMP-2 in periodontal ligament cells by calcium-based biomaterial, Bone Morphogenetic Proteins: New Research, 187-202, 2012.02, Deep caries, severe periodontal diseases, and irreversible trauma cause irretrievable damage to the periodontium, resulting in tooth loss. Many people worldwide are afflicted with these diseases, and thus experience a decreased quality of life. Therefore, researchers have attempted to address this situation by focusing on tissue engineering techniques. The periodontium is mainly composed of two hard tissues and two soft tissues; the former includes alveolar bone and cementum covering the surface of the tooth root, and the latter includes periodontal ligament (PDL) tissue and gingival tissue. In particular, PDL is a specialized, localized connective tissue that bridges the alveolar bone and cementum. Accordingly, complicated growth regulations of these four tissues are required to attain the regeneration of periodontium. Notably, sufficient acquisition of both alveolar bone and PDL tissue is principal in periodontium reconstruction, because severe damage of these tissues elicits extremely difficult regeneration. So far, the potential effects of BMP-2,-4,-6,-7 (OP-1),-12 (GDF7),-13 (GDF6), and-14 (GDF5) on the regeneration of periodontium have been reported. Above all, the promising potency of BMP-2 is well documented in terms of regeneration. In this chapter, we describe the efficacy and prospective roles of BMPs in periodontium regeneration and the potency of calcium-based biomaterial in induction of BMP-2 expression in PDL cells..
50. Tomokiyo A, Maeda H, Fujii S, Monnouchi S, Wada N, Kono K, Koori K, Yamamoto N, Teramatsu Y, Akamine A. , A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival.

, Journal of Cellular Physiology, 2011.07.
51. Hidefumi Maeda, Atsushi Tomokiyo, Fujii Shinsuke, Naohisa Wada, Akifumi Akamine, Promise of periodontal ligament stem cells in regeneration of periodontium, STEM CELL RESEARCH & THERAPY, 10.1186/scrt74, 2, 2011.07.
52. Hidefumi Maeda, Atsushi Tomokiyo, K. Koori, S. Monnouchi, Shinsuke Fujii, Naohisa Wada, K. Kono, N. Yamamoto, T. Saito, A. Akamine, An in vitro evaluation of two resin-based sealers on proliferation and differentiation of human periodontal ligament cells, International Endodontic Journal, 10.1111/j.1365-2591.2010.01845.x, 44, 5, 425-431, 2011.05, [URL], Aim To evaluate the effects of a polymethyl methacrylate resin-based sealer [Superbond sealer (SB)] on the proliferation and osteogenic differentiation of human periodontal ligament cells (HPDLCs) in vitro, compared with a methacrylate resin-based sealer [Epiphany SE sealer (EP)]. Methodology Human periodontal ligament cells were obtained from of healthy third molar teeth of two participants with informed consent. To determine the effects of the eluent from set resin sealers on HPDLCs, the 7-day-washed (washed) or non-washed freshly prepared (fresh) set SB or EP discs were prepared. Cells cultured on these discs were evaluated by the WST-1 proliferation assay and scanning electron microscopy (SEM). The osteogenic differentiation of HPDLCs on washed SB discs was then evaluated by gene expression analysis of osteopontin (OPN) and osteocalcin (OCN) by using quantitative RT-PCR. Results Human periodontal ligament cells exhibited growth on washed SB discs, whereas fresh SB and EP discs and washed EP discs inhibited proliferation of HPDLCs. SEM observation revealed that HPDLCs tightly attached and spread on the surface of washed SB discs, whilst no HPDLCs were observed on the surface of fresh and washed EP discs. Furthermore, HPDLCs significantly upregulated gene expressions of OPN and OCN when cultured on washed SB discs in osteogenic differentiation medium for 2weeks. Conclusions Although Superbond sealer initially exerted cytotoxic effects on HPDLCs, these effects were reduced during washing for 7days compared to EP, which continued to be cytotoxic even though the specimens were washed for the same period of time. Washed Superbond allowed HPDLCs to differentiate into osteogenic cells..
53. isamu hashiguchi, Yoshito Yoshimine, Hidefumi Maeda, Yasuharu GOTO, Naohisa Wada, Fujii Shinsuke, Atsushi Tomokiyo, Kirie Saito, Monnouchi Satoshi, Kono Kiyomi, Akifumi Akamine, An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2010, Fukuoka Igaku Zasshi, 2011.04.
54. Isamu Hashiguchi, Yoshimine Yoshito, Hidefumi Maeda, Yasuharu Gotou, Naohisa Wada, Shinsuke Fujii, Atsushi Tomokiyo, Kirie Saito, Satoshi Monnouchi, Kiyomi Kouno, Hidehiko Okumura, Akifumi Akamine, [An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2010]., Fukuoka Acta Medica, 102, 4, 75-80, 2011.04, An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2010. The results obtained were as follows. 1) Yusho patients complained of tooth pain and periodontal diseases such as gingival swelling, but not of oral pigmentation. 2) 104 patients out of 117 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, 314 teeth out of a total 551 examined teeth showed a periodontal pocket with more than 3 mm in depth. However, it was determined that 57 teeth had a periodontal pocket deeper than 4 mm. 3) Oral pigmentation was observed in 63 patients out of 122 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. The prevalence of oral pigmentation in male patients seemed to be somewhat higher than that in female patients. In addition, the prevalence of oral pigmentation tended to be higher in patients under seventy years old than patients beyond the age of seventy. These results indicated that PCB-related compounds may be responsible for the higher prevalence of both periodontal diseases and oral pigmentation..
55. Maeda H, Tomokiyo A, Koori K, Monnouchi S, Fujii S, Wada N, Kono K, Yamamoto N, Saito T, Akamine A. , An in vitro evaluation of two resin-based sealers on proliferation and differentiation of human periodontal ligament cells.

, Int Endod J. , 2011 May;44(5):425-31. , 44, 5, 425-31, 2011.03.
56. Monnouchi S, Maeda H, Fujii S, Tomokiyo A, Kono K, Akamine A. , The roles of angiotensin II in stretched periodontal ligament cells.

, J Dent Res. , 90, 2, 181-5, 2011.02.
57. S. Monnouchi, Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, K. Kono, A. Akamine, The roles of angiotensin II in stretched periodontal ligament cells, Journal of Dental Research, 10.1177/0022034510382118, 90, 2, 181-185, 2011.02, [URL], The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-Î1 (TGF-Î1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-Î1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-Î1 and ALP..
58. Hidefumi Maeda, Naohisa Wada, Shinsuke Fujii, Atsushi Tomokiyo, Akifumi Akamine, Periodontal ligament stem cells, Stem Cells, 619-636, 2011.
59. Fujii S, Maeda H, Tomokiyo A, Monnouchi S, Hori K, Wada N, Akamine A. , Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line.

, Cell Tissue Res., 342, 2, 233-42, 2010.12.
60. Shinsuke Fujii, Hidefumi Maeda, Atsushi Tomokiyo, Satoshi Monnouchi, Kiyomi Hori, Naohisa Wada, Akifumi Akamine, Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line, Cell and Tissue Research, 10.1007/s00441-010-1037-x, 342, 2, 233-242, 2010.11, [URL], Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions..
61. Maeda H, Nakano T, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Hori K, Akamine A. , Mineral trioxide aggregate induces bone morphogenetic protein-2 expression and calcification in human periodontal ligament cells.

, J Endod., 36, 4, 647-52, 2010.04.
62. Hidefumi Maeda, Tsuguhisa Nakano, Atsushi Tomokiyo, Shinsuke Fujii, Naohisa Wada, Satoshi Monnouchi, Kiyomi Hori, Akifumi Akamine, Mineral Trioxide Aggregate Induces Bone Morphogenetic Protein-2 Expression and Calcification in Human Periodontal Ligament Cells, Journal of Endodontics, 10.1016/j.joen.2009.12.024, 36, 4, 647-652, 2010.04, [URL], Introduction: Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity although the mechanisms remain unclear. We hypothesize that the dissolution of calcium from MTA into the surrounding environment may play an important role in the osteoblastic/cementoblastic differentiation of human periodontal ligament cells (HPLCs). Methods: Two populations of HPLCs were obtained from two patients, respectively, and were cultured in the presence or absence of MTA discs and/or CaCl2 in order to investigate calcium release, calcification activity, calcium-sensing receptor (CaSR) gene expression and bone morphogenetic protein-2 (BMP-2), and BMP-2 receptor protein and gene expression. Results: MTA released a substantial accumulation of calcium (4 mmol/L) within 14 days into culture media. After 4 weeks, the two populations of HPLCs independently exhibited calcification as well as BMP-2 distribution in the vicinity of MTA. HPLCs inherently expressed genes encoding for the CaSR and BMP-2 receptors. Exogenous CaCl2 media supplementation induced CaSR gene expression in HPLCs and calcification and BMP-2 synthesis throughout the entire HPLC cultures, whereas MgCl2 had no effect. Both MTA and CaCl2 stimulated BMP-2 gene expression above that of baseline levels. Conclusion: Here we show the first report showing that HPLCs cocultured directly with MTA up-regulated BMP2 expression and calcification. These results may be through CaSR interactions that were potentially activated by the release of calcium from MTA into the culture environment..
63. 橋口勇、吉嶺嘉人、前田英史、後藤康治、藤井慎介、友清淳、吉田桐枝、西垣奏一郎、門野内聡、堀清美、奥村英彦、赤峰昭文, 油症患者における歯周疾患ならびに口腔内色素沈着の疫学的調査(第7報), 福岡医誌, 100, 5, 111-117, 2009.05.
64. Hashiguchi I, Yoshimine Y, Maeda H, Gotou Y, Fujii S, Tomokiyo A, Yoshida K, Nishigaki S, Monnouch S, Hori K, Okumura H, Akamine A., An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2008., Fukuoka Igaku Zasshi., 100, 5, 111-7, 2009.05.
65. Isamu Hashiguchi, Yoshimine Yoshito, Hidefumi Maeda, Yasuharu Gotou, Shinsuke Fujii, Atsushi Tomokiyo, Kirie Yoshida, Souichiro Nishigaki, Satoshi Monnouch, Kiyomi Hori, Hidehiko Okumura, Akifumi Akamine, An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2008, Fukuoka Acta Medica, 100, 5, 111-117, 2009.05, An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2008. The results obtained were as follows. 1) Yusho patients complained of tooth pain and periodontal diseases such as gingival swelling, gingival bleeding, but not of oral pigmentation. 2) 116 patients out of 148 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, 399 teeth out of a total 710 examined teeth showed a periodontal pocket with more than 3 mm in depth. However, it was determined that 74 teeth had a periodontal pocket deeper than 4 mm. 3) Oral pigmentation was observed in 91 patients out of 155 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. The prevalence of oral pigmentation in male patients seemed to be somewhat higher than that in female patients. In addition, the prevalence of oral pigmentation tended to be higher in younger patients than in elder patients. Pigmentation of the buccal mucosa, lip or palate, however, was observed only in patients beyond the age of fifty. These results indicated that PCB-related compounds may be responsible for the higher prevalence of both periodontal diseases and oral pigmentation..
66. 前田英史、友清淳、藤井慎介、島一也、和田尚久、門野内聡、堀清美、中野嗣久、吉嶺嘉人、赤峰昭文, MTAがヒト歯根膜線維芽細胞に及ぼす影響に関する研究., 日本歯科保存学会誌, 52, 4, 355-362, 2009.04.
67. Fujii S, Maeda H, Wada N, Tomokiyo A, Saito M, Akamine A., Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo., J Cell Physiol., 215(3):743-9., 2008.06.
68. Shinsuke Fujii, Hidefumi Maeda, Naohisa Wada, Atsushi Tomokiyo, Masahiro Saito, Akifumi Akamine, Investigating a clonal human periodontal ligament progenitor/stem cell line in vitro and in vivo, Journal of Cellular Physiology, 10.1002/jcp.21359, 215, 3, 743-749, 2008.06, [URL], The lifespan of the tooth is influenced by the periodontal ligament (PDL), a specialized connective tissue that connects the cementum with the tooth socket bone. Generation of a cell line from PDL progenitor/stem cells would allow development of tissue engineering-based regenerative PDL therapy. However, little is known about the characteristics of PDL progenitor/stem cells because PDL tissue consists of a heterogeneous cell population and there are no pure PDL cell lines. Recently, we succeeded in immortalizing primary human PDL fibroblasts (HPLFs) by transfecting them with SV40 T-antigen and hTERT (Cell Tissue Res 2006; 324: 117-125). In this study, we isolated three clonal cell lines from these immortalized cells (lines 1-4, 1-11, and 1-24) that express RUNX-2, Col1, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and α-SMA mRNA. Immunocytochemical analysis demonstrated that CD146 was expressed in cell lines 1-4 and 1-11 and that STRO-1 was expressed in lines 1-11 and 1-24. Lines 1-4 and 1-11 differentiated into osteoblastic cells and adipocytes when cultured in lineage-specific differentiation media. Four weeks after transplanting cell line 1-11 into immunodeficient mice with β-tricalcium phosphate (β-TCP), the transplant produced cementum/bone-like tissues around the β-TCP. Eight weeks after transplantation, the 1-11 celltransplantformed PDL-like structures on the surface of the β-TCP. These data suggest that cell line 1-11 was derived from a progenitor/stem cell present in the PDL and should be very useful for studying the biology and regeneration of human periodontium..
69. Hashiguchi I, Yoshimine Y, Maeda H, Gotou Y, Ishikawa M, Fujii S, Tomokiyo A, Fukuyama H, Okumura H, Akamine A. , Epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2006., Fukuoka Igaku Zasshi, 98(5):170-5, 2007.05.
70. Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Akamine A., Development of a multipotent clonal human periodontal ligament cell line., Differentiation. , 76(4):337-47, 2008.04.
71. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Naohisa Wada, Kazuya Shima, Akifumi Akamine, Development of a multipotent clonal human periodontal ligament cell line, Differentiation, 10.1111/j.1432-0436.2007.00233.x, 76, 4, 337-347, 2008, [URL], The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1-17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog, as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1-17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 (FGF-R1), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1-17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1-17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1-17 based on its differentiation stage..
72. Isamu Hashiguchi, Yoshimine Yoshito, Hidefumi Maeda, Yasuharu Gotou, Masaki Ishikawa, Shinsuke Fujii, Atsushi Tomokiyo, Hiroshi Fukuyama, Hidehiko Okumura, Akifumi Akamine, Epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2006, Fukuoka Acta Medica, 98, 5, 170-175, 2007.05, An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho in 2006. The results obtained were as follows. (1) 98 patients out of 106 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, 343 teeth out of a total 494 examined teeth showed periodontal pocket with more than 3 mm depth. (2) Oral pigmentation was observed in 61 patients out of 116 patients with Yusho. In this study, gingival pigmentation was most predominant among oral pigmentation. The prevalence of oral pigmentation in male patients seemed to be somewhat higher than that in female patients. In addition, this examination revealed that the patients below the age of sixty had a high prevalence of oral pigmentation. These results indicated that PCB-related compounds may be responsible for the higher prevalence of both periodontal diseases and oral pigmentation..
73. Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. , Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis, Stem Cells., 24(11):2493-503, 2006.11.
74. Koichiro Iohara, Li Zheng, Masataka Ito, Atsushi Tomokiyo, Kenji Matsushita, Misako Nakashima, Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis, Stem Cells, 10.1634/stemcells.2006-0161, 24, 11, 2493-2503, 2006.11, [URL], Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor γ and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration..
75. 野田亮、前田英史、藤井慎介、和田尚久、友清淳、吉嶺嘉人、赤峰昭文, MTA及びSuper-Bondのヒト歯根膜細胞の骨芽細胞様分化に及ぼす影響に関する研究, 日歯内療誌, 27(3):126-131, 2006.03.
76. Nakashima M, Iohara K, Ishikawa M, Ito M, Tomokiyo A, Tanaka T, Akamine A., Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11), Hum Gene Ther, 15(11):1045-53, 2004.11.
77. Misako Nakashima, Koichiro Iohara, Masaki Ishikawa, Masataka Ito, Atsushi Tomokiyo, Takamasa Tanaka, Akifumi Akamine, Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11), Human Gene Therapy, 10.1089/1043034042431164, 15, 11, 1045-1053, 2004.11, [URL], Dental pulp progenitor/stem cells have the capacity to differentiate into odontoblasts and they provide a potential for dentin repair and regeneration by gene therapy. To develop a successful ex vivo gene therapy to induce reparative dentin formation rapidly and effectively after treatment of caries, we developed a three-dimensional pellet culture system of pulp cells electrotransfected with growth/differentiation factor 11 (Gdf11). The viability after electrotransfection was more than 85%, and the efficiency was about 70% as determined by flow cytometry. After 10 days of culture, the total amount of type I and type III collagen was 3-fold higher in the pEGFP-Gdf11-transfected pellet than in the control. Real-time RT-PCR analysis demonstrated that the expression of markers of odontoblast differentiation (alkaline phosphatase, dentin matrix protein 1 [Dmp1], dentin sialophosphoprotein [Dspp], enamelysin, and phosphate-regulating gene with homologies to endopeptidases on X-chromosome [Phex]) was increased in the pEGFP-Gdf11-transfected pellet compared with the control on day 14. On the basis of this in vitro evaluation, an in vivo investigation in the dog was performed. Autogenous transplantation of Gdf11-transfected cells cultured as a pellet on amputated pulp stimulated reparative dentin formation. Thus, Gdf11 gene therapy may be potentially used in endodontic treatment in dentistry..

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