Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Wada Naohisa Last modified date:2021.11.02

Professor / Division of Interdisciplinary Dentistry / Department of Dental Science / Faculty of Dental Science


Papers
1. Hamano S, Tomokiyo A, Hasegawa D, Yuda A, Sugii H, Yoshida S, Mitarai H, Wada N, Maeda H, Functions of beta2-adrenergic receptor in human periodontal ligament cells., J Cell Biochem, 10.1002/jcb.29706, 121, 12, 4798-4808, 2020.12.
2. Inai Y, Nomura Y, Takarada T, Hanada N, Wada N, Risk factors for postoperative pneumonia according to examination findings before surgery under general anesthesia., Clin Oral Investig, 10.1007/s00784-020-03230-7, 24, 10, 3577-3585, 2020.10.
3. Hasegawa D, Hasegawa K, Kaneko H, Yoshida S, Mitarai H, Arima M, Tomokiyo A, Hamano S, Sugii H, Wada N, Kiyoshima T, Maeda H, MEST regulates the stemness of human periodontal ligament stem cells., Stem Cells Int. , 10.1155/2020/9672673. 2020, 9672673 eCollection, 2020.07.
4. Shoko Fujino, Sayuri Hamano, Atsushi Tomokiyo, Tomohiro Itoyama, Daigaku Hasegawa, Hideki Sugii, Shinichiro Yoshida, Ayako Washio, Aoi Nozu, Taiga Ono, Naohisa Wada, Chiaki Kitamura, Hidefumi Maeda, Expression and function of dopamine in odontoblasts., Journal of cellular physiology, 10.1002/jcp.29314, 235, 5, 4376-4387, 2020.05, Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN-3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN-3 cells expressed D1-like and D2-like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN-3 cells, whereas an antagonist of D1-like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2-like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1-like receptors, but not D2-like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis..
5. Osaki A, Sanematsu K, Yamazoe J, Hirose F, Watanabe Y, Kawabata Y, Oike A, Hirayama A, Yamada Y, Iwata S, Takai S, Wada N, Shigemura N, Drinking Ice-Cold Water Reduces the Severity of Anticancer Drug-Induced Taste Dysfunction in Mice., Int J Mol Sci., 10.3390/ijms21238958, 21, 23, 8958, 2020.04.
6. 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, 2020.03, 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..
7. Yuko Inai, Yoshiaki Nomura, Tohru Takarada, Nobuhiro Hanada, Naohisa Wada, Risk factors for postoperative pneumonia according to examination findings before surgery under general anesthesia., Clinical oral investigations, 10.1007/s00784-020-03230-7, 2020.02, OBJECTIVE: This study was performed to determine the risk factors associated with postoperative complications after surgery under general anesthesia according to respiratory function test results and oral conditions. MATERIALS AND METHODS: Preoperative examination data were collected for 471 patients who underwent surgery under general anesthesia at the Medical Hospital of Kyusyu University. Respiratory function tests, oral examinations, and perioperative oral management were performed in all patients. The incidence of and risk factors for postoperative complications were investigated. Classification and regression tree analyses were performed to investigate the risk factors for postoperative complications. RESULTS: Among the 471 patients, 11 developed postoperative pneumonia, 10 developed postoperative respiratory symptoms, and 10 developed postoperative fever. The most important risk factor for pneumonia was edentulism. Age, the Brinkman index, and head and neck surgery were also revealed as important risk factors for pneumonia. The O'Leary plaque control record (initial visit) was an important risk factor for postoperative respiratory symptoms. With respect to postoperative fever, a Hugh-Jones classification of grade > 1 was the most important risk factor; edentulism and a Brinkman index of > 642.5 were also found to be risk factors. CONCLUSION: In addition to respiratory function tests, oral examinations may be important for the prediction of postoperative complications. Additionally, improved oral hygiene may be effective in preventing postoperative respiratory complications. CLINICAL RELEVANCE: Risk factors for postoperative complications should be comprehensively evaluated using both respiratory function tests and oral findings..
8. Shoko Fujino, Sayuri Hamano, Atsushi Tomokiyo, Tomohiro Itoyama, Daigaku Hasegawa, Hideki Sugii, Shinichiro Yoshida, Ayako Washio, Aoi Nozu, Taiga Ono, Naohisa Wada, Chiaki Kitamura, Hidefumi Maeda, Expression and function of dopamine in odontoblasts, Journal of cellular physiology, 10.1002/jcp.29314, 235, 5, 4376-4387, 2020.01, Dopamine (DA) is produced from tyrosine by tyrosine hydroxylase (TH). A recent study has reported that DA promotes the mineralization of murine preosteoblasts. However, the role of DA in odontoblasts has not been examined. Therefore, in this investigation, we researched the expression of TH and DA in odontoblasts and the effects of DA on the differentiation of preodontoblasts (KN-3 cells). Immunostaining showed that TH and DA were intensely expressed in odontoblasts and preodontoblasts of rat incisors and molars. KN-3 cells expressed D1-like and D2-like receptors for DA. Furthermore, DA promoted odontoblastic differentiation of KN-3 cells, whereas an antagonist of D1-like receptors and a PKA signaling blocker, inhibited such differentiation. However, antagonists of D2-like receptors promoted differentiation. These results suggested that DA in preodontoblasts and odontoblasts might promote odontoblastic differentiation through D1-like receptors, but not D2-like receptors, and PKA signaling in an autocrine or paracrine manner and plays roles in dentinogenesis..
9. Osada K, Yugawa A, Yamazoe J, Wada N, Oral Management Due to Denture Adjustment in a Frail Patient with Spinocerebellar Degeneration Contributed to Effective Rehabilitation, 老年歯科医学, 35, 1, 61-69, 2020.01.
10. Shinsuke Fujii, Kengo Nagata, Shinji Matsumoto, Kenichi Kouhashi, Akira Kikuchi, Yoshinao Oda, Tamotsu Kiyoshima, Naohisa Wada, Wnt/β-catenin signaling, which is activated in odontomas, reduces Sema3A expression to regulate odontogenic epithelial cell proliferation and tooth germ development, Scientific reports, 10.1038/s41598-019-39686-1, 9, 1, 2019.12.
11. Atsushi Tomokiyo, Naohisa Wada, Hidefumi Maeda, Periodontal Ligament Stem Cells: Regenerative Potency in Periodontium., Stem cells and development, 10.1089/scd.2019.0031, 28, 15, 974-985, 2019.08, Periodontium is consisted of root cementum, bone lining the tooth socket, gingiva facing the tooth, and periodontal ligament (PDL). Its primary functions are support of the tooth and protection of tooth, nerve, and blood vessels from injury by mechanical loading. Severe periodontitis induces the destruction of periodontium and results in a significant cause of tooth loss among adults. Unfortunately, conventional therapies such as scaling and root planning are often only palliative. Therefore, the ultimate goal of the treatment for periodontitis is to restore disrupted periodontium to its original shape and function. Tissue engineering refers to the process of combining cells, scaffolds, and signaling molecules for the production of functional tissues to restore, maintain, and improve damaged organs. The discovery of periodontal ligament stem cells (PDLSCs) highlighted the possibility for development of tissue engineering technology-based therapeutics for disrupted periodontium. PDLSCs are a kind of somatic stem cells that show potential to differentiate into multiple cell types and undergo robust clonal self-renewal. Therefore, PDLSCs are considered a highly promising stem cell population for regenerative therapy in periodontium; however, their rarity prevents the progression of basic and clinical researches. In this review, we summarize recent research advancement and accumulated information regarding the self-renewal capacity, multipotency, and immunomodulatory effect of PDLSCs, as well as their contribution to repair and regeneration of periodontium and other tissues. We also discuss the possibility of PDLSCs for clinical application of regenerative medicine and provide an outline of the genetic approaches to overcome the issue about the rarity of PDLSCs..
12. 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, 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..
13. 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, 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..
14. Wnt/β-catenin signaling, which is activated in odontomas, reduces Sema3A expression to regulate odontogenic epithelial cell proliferation and tooth germ development..
15. Yuriko Harada, Kenji Takeuchi, Michiko Furuta, Akihiko Tanaka, Shunichi Tanaka, Naohisa Wada, Yoshihisa Yamashita, Gender-dependent associations between occupational status and untreated caries in Japanese adults, Industrial health, 10.2486/indhealth.2018-0062, 56, 6, 539-544, 2018.11, The aim of this study was to examine whether the presence of untreated caries is different across occupational status among Japanese adults. This was a cross-sectional survey of 1,342 individuals (990 males and 352 females) aged 40-64 yr who underwent medical and dental checkups at a healthcare center in 2011. Oral examination was performed by a dentist and the presence of untreated caries was defined as having at least one untreated decayed tooth. Data regarding current occupational status were obtained using a self-administered questionnaire; the participants were classified into five groups: professionals and managers, clerical and related workers, service and salespersons, agricultural, forestry, and fishery workers, and homemakers and unemployed. Gender-specific odds ratios (ORs) and 95% confidence intervals (CIs) of occupational status for the presence of untreated caries were estimated using logistic regression. After adjusting for potential confounders, female professionals and managers (OR=3.51, 95% CI=1.04-11.87) and service and salespersons (OR=5.29, 95% CI=1.39-20.11) had greater risks of the presence of untreated caries than female homemakers and unemployed. However, this tendency was not observed among males. In conclusion, there was a significant difference in risk of the presence of untreated caries by occupational status among females..
16. Yuriko Harada, Kenji Takeuchi, Michiko Furuta, Akihiko Tanaka, Shunichi Tanaka, Naohisa Wada, Yoshihisa Yamashita, Gender-dependent associations between occupational status and untreated caries in Japanese adults., Industrial health, 10.2486/indhealth.2018-0062, 56, 6, 539-544, 2018.11, The aim of this study was to examine whether the presence of untreated caries is different across occupational status among Japanese adults. This was a cross-sectional survey of 1,342 individuals (990 males and 352 females) aged 40-64 yr who underwent medical and dental checkups at a healthcare center in 2011. Oral examination was performed by a dentist and the presence of untreated caries was defined as having at least one untreated decayed tooth. Data regarding current occupational status were obtained using a self-administered questionnaire; the participants were classified into five groups: professionals and managers, clerical and related workers, service and salespersons, agricultural, forestry, and fishery workers, and homemakers and unemployed. Gender-specific odds ratios (ORs) and 95% confidence intervals (CIs) of occupational status for the presence of untreated caries were estimated using logistic regression. After adjusting for potential confounders, female professionals and managers (OR=3.51, 95% CI=1.04-11.87) and service and salespersons (OR=5.29, 95% CI=1.39-20.11) had greater risks of the presence of untreated caries than female homemakers and unemployed. However, this tendency was not observed among males. In conclusion, there was a significant difference in risk of the presence of untreated caries by occupational status among females..
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.
18. 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, 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..
19. 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, 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..
20. 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, 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..
21. 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, 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..
22. 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, 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..
23. 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.
24. 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, 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..
25. Suguru Serita, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Shinichiro Yoshida, Hiroyuki Mizumachi, Hiromi Mitarai, Satoshi Monnouchi, Naohisa Wada, Hidefumi Maeda, Transforming growth factor-β-induced gene product-h3 inhibits odontoblastic differentiation of dental pulp cells., Archives of oral biology, 10.1016/j.archoralbio.2017.02.018, 78, 135-143, 2017.06, 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..
26. 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, 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. © 2016 Wiley Periodicals, Inc..
27. Suguru Serita, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Shinichiro Yoshida, H. Mizumachi, Hiromi Mitarai, S. Monnouchi, 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, 2017.02.
28. H. Mizumachi, Shinichiro Yoshida, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, Asuka Yuda, Hideki Sugii, Suguru Serita, Hiromi Mitarai, K. 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, 2017.02.
29. Kana Hasegawa, Hiroko Wada, Kengo Nagata, Hiroaki Fujiwara, Naohisa Wada, Hirotaka Someya, Yurie Mikami, Hidetaka Sakai, Tamotsu Kiyoshima, Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development., Journal of molecular histology, 10.1007/s10735-016-9680-5, 47, 4, 375-87, 2016.08, Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the inner enamel epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation..
30. Shinichiro Yoshida, N. Yamamoto, Naohisa Wada, Atsushi Tomokiyo, Daigaku Hasegawa, Sayuri Hamano, H. Mitarai, S. Monnouchi, A. Yuda, Hidefumi Maeda, GDNF from Human Periodontal Ligament Cells Treated with Proinflammatory Cytokines Promotes Neurocytic Differentiation of PC12 Cells., J Cell Biochem, 10.1002/jcb.25662, 118, 4, 699-708, 2016.07.
31. Shinichiro Yoshida, Naohisa Wada, Daigaku Hasegawa, H. Miyaji, H. Mitarai, Atsushi Tomokiyo, Sayuri Hamano, Hidefumi Maeda, Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells., J Dent Res, 10.1177/0022034516653085, 95, 11, 1282-1290, 2016.06.
32. Kana Hasegawa, Hiroko Wada, Kengo Nagata, Hiroaki Fujiwara, Naohisa Wada, Someya H, Mikami Y, Sakai Hidetaka, Tamotsu Kiyoshima, Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development., J Mol Histol, 10.1007/s10735-016-9680-5, 47, 4, 375-387, 2016.05.
33. Monnouchi Satoshi, 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, in press, 2016.01.
34. Daigaku Hasegawa, Naohisa Wada, Hidefumi Maeda, S.Yoshida, H. Mitarai, Atsushi Tomokiyo, Monnouchi Satoshi, S. Hamano, A. Yuda, Akifumi Akamine, Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells through TGFβ1-mediated Upregulation of Periostin Expression., J Cell Physiol, 230, 11, 2647-2660, 2015.11.
35. Hasegawa D, Wada N, Maeda H, Yoshida S, Mitarai H, Tomokiyo A, Monnouchi S, Hamano S, Yuda A, Akamine A, Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells Through TGFβ1-Mediated Upregulation of Periostin Expression., Journal of cellular physiology, 10.1002/jcp.24950, 230, 11, 2647-60, 2015.11.
36. 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, 150-9, 2015.01, 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..
37. 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, 10.14670/HH-29.1249, 29, 10, 1249-62, 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..
38. 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, 2014.09, 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..
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, 633-43, 2014.09, 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, 2225-36, 2014.09, 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, 707-18, 2014.09, 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. Zakaria MN, Toru Takeshita, Yukie Shibata, Hidefumi Maeda, Naohisa Wada, Akifumi Akamine, Yoshihisa Yamashita, Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis., Int Endod J, in press, 2014.08.
43. Asuka Yuda, Hidefumi Maeda, Fujii Shinsuke, Monnouchi Satoshi, Naohide Yamamoto, Naohisa Wada, Koori Katsuaki, 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., J Cell Physiol, Epub ahead of print, 2014.06.
44. Hideki Sugii, Hidefumi Maeda, Atsushi Tomokiyo, Naohide Yamamoto, Naohisa Wada, Koori Katsuaki, Daigaku Hasegawa, Sayuri Hamano, Asuka Yuda, Monnouchi Satoshi, Akifumi Akamine, Effects of Activin A on the phenotypic properties of human periodontal ligament cells., Bone, in press, 2014.06.
45. Monnouchi Satoshi, Hidefumi Maeda, Asuka Yuda, Sayuri Hamano, Naohisa Wada, Atsushi Tomokiyo, Koori Katsuaki, Hideki Sugii, Suguru Serita, Akifumi Akamine, Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells., J Periodontal Res, in press, 2014.06.
46. Menicanin D, Mrozik M, Naohisa Wada, Marino V, Shi S, Bartold PM, Gronthos S., Periodontal Ligament Derived Stem Cells Exhibit the Capacity for Long-Term Survival, Self-Renewal and Regeneration of Multiple Tissue Types in Vivo. , Stem Cells Dev, 23, 9, 1001-1011, 2014.05.
47. Koori Katsuaki, Hidefumi Maeda, Fujii Shinsuke, Atsushi Tomokiyo, G 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 Tissue Res, Epub ahead of print, 2014.05.
48. 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 Tissue Res, 2014.05.
49. Danijela Menicanin, Krzysztof Marek Mrozik, Naohisa Wada, Victor Marino, Songtao Shi, P Mark Bartold, Stan Gronthos, Periodontal-ligament-derived stem cells exhibit the capacity for long-term survival, self-renewal, and regeneration of multiple tissue types in vivo., Stem cells and development, 10.1089/scd.2013.0490, 23, 9, 1001-11, 2014.05, Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)-labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo-expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam(®) scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model..
50. Naohisa Wada, Hidefumi Maeda, Hasegawa D, Gronthos S, Bartold PM, Menicanin D, Fujii S, Yoshida D, Tomokiyo A, Monnouchi Satoshi, Akifumi Akamine, Semaphorin 3A induces mesenchymal stem-like properties in human periodontal ligament cells., Stem Cells Dev, in press, 2014.02.
51. Periodontal tissue engineering: defining the triad..
52. Krzysztof Marek Mrozik, Naohisa Wada, Victor Marino, Ward Richter, Songtao Shi, Donna L Wheeler, Stan Gronthos, P Mark Bartold, Regeneration of periodontal tissues using allogeneic periodontal ligament stem cells in an ovine model., Regenerative medicine, 10.2217/rme.13.66, 8, 6, 711-23, 2013.11, AIM: To investigate the capacity of allogeneic periodontal ligament stem cells (PDLSCs) to regenerate periodontal tissues using an ovine periodontal defect model. MATERIALS & METHODS: Surgically created zero-wall dehiscence periodontal defects created in Merino sheep were filled with 1 × 10(7) allogeneic PDLSCs attached to Gelfoam(®), Gelfoam alone or left untreated. After 4 weeks, histological analysis was performed to assess periodontal regeneration. RESULTS: Allogeneic PDLSCs were well tolerated by recipient animals. The mean area of new alveolar bone was significantly greater in the PDLSC + Gelfoam treatment group compared with the defect-alone group. The PDLSC + Gelfoam and Gelfoam-only treatment groups displayed significantly greater length of new cementum and percentage of cementum regrowth compared with the defect-alone group. New Sharpey's fibers were generally more organized and significantly thicker within the PDLSC + Gelfoam treatment group. The PDLSC + Gelfoam treatment group also showed a trend of increased Sharpey's fiber attachment length compared with the Gelfoam-only and defect-alone groups. CONCLUSION: These studies support the potential use of allogeneic PDLSC preparations as viable therapies for periodontal regeneration in the clinical setting..
53. Naohisa Wada, Stan Gronthos, P Mark Bartold, Immunomodulatory effects of stem cells., Periodontology 2000, 10.1111/prd.12024, 63, 1, 198-216, 2013.10, Adult-derived mesenchymal stem cells have received considerable attention over the past two decades for their potential use in tissue engineering, principally because of their potential to differentiate into multiple stromal-cell lineages. Recently, the immunomodulatory properties of mesenchymal stem cells have attracted interest as a unique property of these cells that may be harnessed for novel therapeutic approaches in immune-mediated diseases. Mesenchymal stem cells have been shown to inhibit the proliferation of activated T-cells both in vitro and in vivo but to stimulate T-regulatory cell proliferation. Mesenchymal stem cells are also known to be weakly immunogenic and to exert immunosuppressive effects on B-cells, natural killer cells, dendritic cells and neutrophils through various mechanisms. Furthermore, intravenous administration of allogeneic mesenchymal stem cells has shown a marked suppression of host immune reactions in preclinical animal models of large-organ transplant rejection and in various autoimmune- and inflammatory-based diseases. Some clinical trials utilizing human mesenchymal stem cells have also produced promising outcomes in patients with graft-vs.-host disease and autoimmune diseases. Mesenchymal stem cells identified from various dental tissues, including periodontal ligament stem cells, also possess multipotent and immunomodulatory properties. Hence, dental mesenchymal stem cells may represent an alternate cell source, not only for tissue regeneration but also as therapies for autoimmune- and inflammatory-mediated diseases. These findings have elicited interest in dental tissue mesenchymal stem cells as alternative cell sources for modulating alloreactivity during tissue regeneration following transplantation into human leukocyte antigen-mismatched donors. To examine this potential in periodontal regeneration, future work will need to assess the capacity of allogeneic periodontal ligament stem cells to regenerate periodontal ligament in animal models of periodontal disease. The present review describes the immunosuppressive effects of mesenchymal stem cells on various types of immune cells, the potential mechanisms through which they exert their mode of action and the preclinical animal studies and human clinical trials that have utilized mesenchymal stem cells, including those populations originating from dental structures..
54. Mrozik K, Naohisa Wada, Marino V, Richter W, Shi S, Wheeler DL, Gronthos S, Bartold PM, Regeneration of periodontal tissues using allogeneic periodontal ligament stem cells in an ovine model., Regen Med, 8, 6, 711-723, 2013.06.
55. Kono Kiyomi, Hidefumi Maeda, Fujii Shinsuke, Atsushi Tomokiyo, Yamamoto N, Naohisa Wada, Monnouchi Satoshi, Teramatsu Y, Hamano S, Koori K, 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 Tissue Res, 352, 2, 249-263, 2013.05.
56. Kono K, Maeda H, Fujii S, Tomokiyo A, Yamamoto N, Wada N, Monnouchi S, Teramatsu Y, Hamano S, Koori K, Akamine A, 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, 249-63, 2013.05.
57. Teramachi J, Kukita A, Qu P, naohisa wada, Li YJ, Toshio Kukita, Adenosine blocks aminopterin-induced suppression of osteoclast differentiation., J Bone Miner Metab, 31, 1, 64-70, 2013.01.
58. Junpei Teramachi, Akiko Kukita, Pengfei Qu, Naohisa Wada, Yin-Ji Li, Seiji Nakamura, Toshio Kukita, Adenosine blocks aminopterin-induced suppression of osteoclast differentiation., Journal of bone and mineral metabolism, 10.1007/s00774-012-0388-7, 31, 1, 64-70, 2013.01, To search cell surface molecules involved in the regulation of osteoclastogenesis, especially in fusion process, it is one powerful approach to obtain monoclonal antibodies bearing ability to block formation of multinucleated osteoclasts. Ideally, direct bio-assay of hybridoma supernatants is quite convenient to screen monoclonal antibodies of interest from numerous culture wells. However, addition of hybridoma supernatant containing hypoxanthine-aminopterin-thymidine (HAT), components of the selection medium, to whole bone marrow cultures strikingly suppressed osteoclastogenesis. Here we clarified aminopterin is the responsible component in HAT medium to inhibit osteoclastogenesis. Methotrexate (MTX), mono-methylated aminopterin, showed similar suppressive effect on osteoclastogenesis. When bone marrow cells were cultured in the presence of all nucleosides, aminopterin and MTX-induced suppression of osteoclastogenesis was abrogated. Among four nucleosides only adenosine canceled aminopterin-induced suppression of osteoclastogenesis. Direct bio-assay of hybridoma supernatant containing HAT selection medium is now available to screen monoclonal antibodies if adenosine-containing culture medium was utilized for evaluating osteoclastogenesis..
59. Naohisa Wada, Hideaki Fujii, Koji Koyano, Shigeto Hirayama, Takashi Iwai, Toru Nemoto, Hiroshi Nagase, Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons and their pharmacologies. 3: synthesis of novel triplet drugs with the bis(epoxymethano) or bis(dimethylepoxymethano) structure (double-capped triplet)., Bioorganic & medicinal chemistry letters, 10.1016/j.bmcl.2012.10.023, 22, 24, 7551-4, 2012.12, Novel double-capped triplet drugs, which have one pharmacophore unit and two epoxymethano or dimethylepoxymethano structures (termed cap or diMe-cap structures, respectively) were synthesized. Key intermediate oxazoline 16 derived from acetone enabled the effective synthesis of double-capped triplets. SYK-134 (7a) and SYK-135 (8a) with N-cyclopropylmethyl substituent and cap structures showed selectivities for the κ opioid receptor. On the other hand, the N-Me series exhibited selectivities for the μ opioid receptor. The double-capped triplet drugs with diMe-cap structures preferred the μ receptor independently of their N-substituents. SYK-385 (19b), one of the μ-selective double-capped triplet drugs, showed the highest selectivity for the μ receptor among the reported μ-selective nonpeptide ligands..
60. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohisa Wada, Kiyomi Hori, Katsuaki Koori, 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, 3093-103, 2012.10, It is well known that the aryl hydrocarbon receptor (AhR) is involved in the toxicity of halogenated aromatic hydrocarbons (HAH) and polycyclic aromatic hydrocarbons (PAH). Recent experiments have shown the induction of impaired tooth and hard-tissue formation by AhR pathway activation, however, the effect on periodontal ligament (PDL) tissue remains unclear. Here, we investigated the effects of benzo(a)pyrene (BaP), a member of PAH, on the extracellular matrix (ECM) remodeling-related molecules, collagen type I (COL-I), matrix metalloproteinase-1 (MMP-1), alpha-smooth muscle actin (α-SMA) expression, and apoptosis in two different human periodontal ligament cells (HPDLCs). The transduction of AhR from the cytoplasm to the nucleus and the increase of AhR-responsive genes; that is, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and aryl-hydrocarbon receptor repressor (AhRR), expression was induced by BaP exposure in both HPDLCs. BaP treatment significantly enhanced MMP-1 mRNA expression and MMP-1 protein production, while markedly suppressing COL-I and a-SMA mRNA expression in both HPDLCs. Furthermore, these BaP-treated HPDLCs fell into apoptotic cell death as evidenced by induction in annexin V and caspase-3/7 staining and reduction of total cell number and Bcl-2 mRNA expression. Thus, BaP exposure altered the expression of ECM-related molecules and induced apoptosis in HPDLCs through activation of the AhR pathway. Overactivity of the AhR pathway may induce an inappropriate turnover of PDL tissue via disordered ECM remodeling and apoptosis in PDL cells..
61. Yamamoto N, Maeda H, Tomokiyo A, Fujii S, Wada N, Monnouchi S, Kono K, Koori K, Teramatsu Y, Akamine A, Expression and effects of glial cell line-derived neurotrophic factor on periodontal ligament cells., J Clin Periodontol, 39, 6, 556-564, 2012.06.
62. Naohide Yamamoto, Hidefumi Maeda, Atsushi Tomokiyo, Shinsuke Fujii, Naohisa Wada, Satoshi Monnouchi, Kiyomi Kono, Katsuaki Koori, 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, 556-64, 2012.06, AIM: To investigate Glial cell line-derived neurotrophic factor (GDNF) expression in normal and wounded rat periodontal ligament (PDL) and the effects of GDNF on human PDL cells (HPDLCs) migration and extracellular matrix expression in HPDLCs. MATERIAL AND METHODS: The expression of GDNF and GDNF receptors was examined by immunocyto/histochemical analyses. Gene expression in HPDLCs treated with GDNF, interleukin-1 beta (IL-1β), or tumour necrosis factor-alpha (TNF-α) was quantified by quantitative RT-PCR (qRT-PCR). In addition, we examined the migratory effect of GDNF on HPDLCs. RESULTS: GDNF was expressed in normal rat PDL and cultured HPDLCs. HPDLCs also expressed GDNF receptors. In wounded rat PDL, GDNF expression was up-regulated. QRT-PCR analysis revealed that IL-1β and TNF-α significantly increased the expression of GDNF in HPDLCs. Furthermore, GDNF induced migration of HPDLCs, which was blocked by pre-treatment with the peptide including Arg-Gly-Asp (RGD) sequence, or neutralizing antibodies against integrin αVβ3 or GDNF. Also, GDNF up-regulated expression of bone sialoprotein (BSP) and fibronectin in HPDLCs. CONCLUSIONS: GDNF expression is increased in rat wounded PDL tissue and HPDLCs treated with pro-inflammatory cytokines. GDNF enhances the expression of BSP and fibronectin, and migration in an RGD-dependent manner via the integrin αVβ3. These findings suggest that GDNF may contribute to wound healing in PDL tissue..
63. naohisa wada, Hidefumi Maeda, Kono Kiyomi, Atsushi Tomokiyo, Monnouchi Satoshi, Katsuaki Koori, Naohide Yamamoto, Yoko Teramatsu, Akifumi Akamine, A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival., J Cell Physiol, 227, 5, 2040-2050, 2012.05.
64. Atsushi Tomokiyo, Hidefumi Maeda, Fujii Shinsuke, Monnouchi Satoshi, naohisa wada, Kono Kiyomi, Koori K, Yamamoto N, Teramatsu Y, Akifumi Akamine, Alternation of extracellular matrix remodeling and apoptosis by activation of the aryl hydrocarbon receptor pathway in human periodontal ligament cells., J Cell Biochem, 113, 10, 3093-3103, 2012.05.
65. 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, 2040-50, 2012.05, 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 50 pg/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..
66. 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, 44, 5, 425-431, 2011.07.
67. Hidefumi Maeda, Atsushi Tomokiyo, Shinsuke Fujii, Naohisa Wada, Akifumi Akamine, Promise of periodontal ligament stem cells in regeneration of periodontium., Stem cell research & therapy, 10.1186/scrt74, 2, 4, 33-33, 2011.07, A great number of patients around the world experience tooth loss that is attributed to irretrievable damage of the periodontium caused by deep caries, severe periodontal diseases or irreversible trauma. The periodontium is a complex tissue composed mainly of two soft tissues and two hard tissues; the former includes the periodontal ligament (PDL) tissue and gingival tissue, and the latter includes alveolar bone and cementum covering the tooth root. Tissue engineering techniques are therefore required for regeneration of these tissues. In particular, PDL is a dynamic connective tissue that is subjected to continual adaptation to maintain tissue size and width, as well as structural integrity, including ligament fibers and bone modeling. PDL tissue is central in the periodontium to retain the tooth in the bone socket, and is currently recognized to include somatic mesenchymal stem cells that could reconstruct the periodontium. However, successful treatment using these stem cells to regenerate the periodontium efficiently has not yet been developed. In the present article, we discuss the contemporary standpoints and approaches for these stem cells in the field of regenerative medicine in dentistry..
68. Teramachi J, Kukita A, Li YJ, Ushijima Y, Ohkuma H, Wada N, Watanabe T, Nakamura S, Kukita T, Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis, Lab Invest, 91, 5, 719-731, 2011.05.
69. Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM, Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts., J Periodontal Res, 46, 4, 438-447, 2011.05.
70. Junpei Teramachi, Akiko Kukita, Yin-Ji Li, Yuki Ushijima, Hiroshi Ohkuma, Naohisa Wada, Toshiyuki Watanabe, Seiji Nakamura, Toshio Kukita, Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis., Laboratory investigation; a journal of technical methods and pathology, 10.1038/labinvest.2011.9, 91, 5, 719-31, 2011.05, Methotrexate (MTX) is widely utilized for the treatment of patients with rheumatoid arthritis (RA); however, recent observation of the MTX-resistant patients proposed some difficulty in MTX-dependent therapeutic approach for RA. To access cellular events related to MTX resistance in RA in respect to inflammatory bone destruction, we investigated on an involvement of the potent inflammatory mediator adenosine in the regulation of osteoclastogenesis and inflammatory bone destruction. In rats with adjuvant-induced arthritis (AA rats), MTX efficiently suppressed bone destruction when it was administrated within 3 days after adjuvant injection, while it could not suppress inflammatory bone destruction if MTX was injected at the time of onset of inflammation (at day 10 after adjuvant injection). Time-course change in the level of plasma adenosine of AA rats was estimated by use of high-performance liquid chromatography and elucidated that adenosine level was markedly elevated till 10 days after adjuvant injection. In vitro bone marrow culture system for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis in a stromal cell-dependent manner. This MTX-induced suppression of osteoclastogenesis was abrogated by the addition of adenosine. MTX suppressed the expression of mRNA for the receptor activator NF-κB ligand (RANKL), but it did not suppress the expression of osteoprotegerin (OPG). The addition of MTX and adenosine together markedly suppressed the level of OPG expression. Abolishment of MTX action by adenosine was significantly blocked by MRS1754, a highly selective antagonist for the A(2b) adenosine receptor (A(2b)AR), but not by caffeine, an antagonist for A₁, A(2a), A₃ AR (A₁AR, A(2a)AR, and A₃AR), which suggests that adenosine acts through A(2b)AR. Immunohistochemical studies showed abundant expression of A(2b)AR in cells localized in the bone-bone marrow boundary of the distal tibia in AA rats but not in control rats. When adenosine was injected in the ankle joints of MTX-treated AA rats, the suppressive effects of MTX on bone destruction was abolished. The current data therefore suggest that upregulation of adenosine production abolished the suppressive effect of MTX on osteoclastic bone destruction. Involvement of the adenosine-A(2b)AR system may explain MTX resistance in RA..
71. 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, 44, 5, 425-431, 2011.05.
72. Naohisa Wada, Peter Mark Bartold, Stan Gronthos, Human foreskin fibroblasts exert immunomodulatory properties by a different mechanism to bone marrow stromal/stem cells., Stem cells and development, 10.1089/scd.2010.0246, 20, 4, 647-59, 2011.04, Human bone marrow mesenchymal stromal/stem cells (BMSCs) have been reported to possess immunomodulatory functions with the capacity to suppress immune reactions partly mediated by immunosuppressive factors, indoleamine 2,3-dioxygenase and nitric oxide, and suggested to be potentially applicable for therapeutic use. More recently, other fibroblast-like cells have been reported to possess similar properties. Here we demonstrate that human foreskin fibroblasts (HFFs) express an MSC-like immunophenotype and possess immunosuppressive properties similar to BMSCs but lack the capacity for osteogenic and adipogenic differentiation. HFFs suppressed human peripheral blood mononuclear cells (PBMC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction comparable to BMSCs. However, HFFs showed undetectable levels of indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression, in contrast to BMSCs when cocultured with activated PBMCs. To identify HFF specific immunosuppressive factors, we performed array profiling of common cytokines expressed by HFFs and BMSCs alone or when cocultured with activated PBMCs. Real-time polymerase chain reaction analysis confirmed that multiple factors were upregulated in HFFs cocultured with activated PBMCs compared with HFFs alone or BMSCs cultured under the same conditions. Functional assays identified interferon-α as the major immunosuppressive mediator expressed by HFFs. These results suggest that the HFFs possess immunosuppressive properties, which are mediated by alternate mechanisms to that reported for BMSCs..
73. 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 and tissue research, 10.1007/s00441-010-1037-x, 342, 2, 233-42, 2010.11.
74. 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, 2010.10.
75. Wada N, Bartold PM, Gronthos S, Human foreskin fibroblasts exert immunomodulatory properties by a different mechanism to bone marrow mesenchymal stem cells, Stem Cells Dev, 20, 4, 647-659, 2010.09.
76. 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, 647-652, 2010.04.
77. 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, 647-52, 2010.04, 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 CaCl(2) 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 CaCl(2) media supplementation induced CaSR gene expression in HPLCs and calcification and BMP-2 synthesis throughout the entire HPLC cultures, whereas MgCl(2) had no effect. Both MTA and CaCl(2) 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..
78. Naohisa Wada, Danijela Menicanin, Songtao Shi, P Mark Bartold, Stan Gronthos, Immunomodulatory properties of human periodontal ligament stem cells., Journal of cellular physiology, 10.1002/jcp.21710, 219, 3, 667-76, 2009.06, Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell-based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non-cell contact dependent suppression of PBMNC proliferation in co-cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN-gamma partially suppressed PBMNC proliferation when compared to CMs without IFN-gamma stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF-beta1, hepatocyte growth factor (HGF) and indoleamine 2, 3-dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN-gamma. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667-676, 2009. (c) 2009 Wiley-Liss, Inc..
79. Wada N, Menicanin D, Shi S, Bartold PM, Gronthos S, Immunomodulatory properties of human periodontal ligament stem cells, J Cell Physiol, 219, 667-676, 2009.01.
80. 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, 743-9, 2008.06, 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, Col I, ALP, OPN, OCN, RANKL, OPG, scleraxis, periostin, Col XII, and alpha-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 beta-tricalcium phosphate (beta-TCP), the transplant produced cementum/bone-like tissues around the beta-TCP. Eight weeks after transplantation, the 1-11 cell transplant formed PDL-like structures on the surface of the beta-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..
81. Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Akamine A, Development of a multipotent clonal human periodontal ligament cell line. Differentiation, Differentiation, 76, 4, 337-347, 2008.04.
82. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Naohisa Wada, Kazuya Shima, Akifumi Akamine, Development of a multipotent clonal human periodontal ligament cell line., Differentiation; research in biological diversity, 10.1111/j.1432-0436.2007.00233.x, 76, 4, 337-47, 2008.04, 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..
83. 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, 743-749, 2008.03.
84. SEM images of root canal dentin irrigated with EDTA and NaOCl -Comparison with ultrasonic irrigation-.
85. Effects of root-end filling materials on the osteoblast-like differentiation of human periodontal ligament cells.
86. Fujii S, Maeda H, Wada N, Kano Y, Akamine A, Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer., Cell and Tissue Research, vol.324(1):pp117-125, 2006.04.
87. Shinsuke Fujii, Hidefumi Maeda, Naohisa Wada, Yoshio Kano, Akifumi Akamine, Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer., Cell and tissue research, 10.1007/s00441-005-0101-4, 324, 1, 117-25, 2006.04, The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL..
88. Hidefumi Maeda, Naohisa Wada, Shinsuke Fujii, Akifumi Akamine, Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified boiled rat bone., Cell and tissue research, 10.1007/s00441-004-1052-x, 320, 1, 135-40, 2005.04, We have been studying the potential of human fibroblastic cells (HFC) from periapical granulation tissue to form a calcified matrix. Recently, we reported that inflamed periapical granulation tissue contains osteogenic cells. In the present study, we tested the hypothesis that HFC, cultured with decalcified bone (DB) of rat, might form much greater calcified matrices than with rat decalcified boiled bone (DBB), which was originally prepared as a negative control. HFC were cultured with DB or DBB in the presence or absence of 2 mM beta-glycerophosphate (beta-GP) and 50 microg/ml ascorbic acid. After six weeks of culture, a number of von Kossa-positive globular structures were unexpectedly observed inside DBB, but not DB. Without HFC, such structures were never seen in DBB incubated with 2 mM beta-GP and 50 microg/ml ascorbic acid. DB cultured with HFC under the same conditions did not show these structures. Electron-microscopic observation revealed that matrix vesicles aggregated on collagen fibrils around globular structures in DBB. Energy dispersive X-ray microanalysis confirmed that these structures were calcified matrices composed of calcium and phosphate. These results suggest that human periapical granulation tissue contains cells responsible for the formation of calcified matrices in DBB, and that DBB could serve as an excellent scaffold for the calcification of HFC, rather than DB..
89. Hisayuki Nomiyama, Kimie Egami, Naohisa Wada, Kazuko Tou, Madoka Horiuchi, Hiromi Matsusaki, Retsu Miura, Osamu Yoshie, Toshio Kukita, Identification of genes differentially expressed in osteoclast-like cells., Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 25, 4, 227-31, 2005.04, Homeostasis of the skeletal system is maintained by a balance between bone formation and resorption. The receptor activator of NF-kappaB ligand (RANKL) induces the differentiation of bone-resorbing cells, osteoclasts. To identify genes regulated during osteoclast differentiation, we constructed a subtraction cDNA library using a mouse RAW264 macrophage cell line that differentiates into osteoclast-like multinucleated cells after treatment with RANKL. Northern blot analysis showed that RANKL treatment upregulated expression of 17 genes. Among these were the genes for five H(+)-ATPase subunits, two chemokines, and the osteoclast marker cathepsin K. In addition, a mouse homolog of human dendritic cell (DC)-specific transmembrane protein (DCSTAMP), whose function in osteoclastogenesis was recently revealed, was also included in the induced genes. Characterization of these inducible genes will provide an insight into the biology of osteoclasts and the mechanism of bone-related diseases..
90. Maeda H, Wada N, Fujii S, Akamine A, Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified and boiled rat bone., Cell and Tissue Research, 10.1007/s00441-004-1052-x, 320, 1, 135-140, vol.320: pp135-140, 2005.01.
91. Nomiyama H, Egami K, Wada N, Tou K, Horiuchi M, Matsusaki H, Miura R, Yoshie O, Kukita T, Identification of genes differentially expressed in osteoclast-like cells., Journal of Interferon and Cytokine Research, 10.1089/jir.2005.25.227, 25, 4, 227-231, vol.25: pp227-231, 2005.01.
92. Toshio Kukita, Naohisa Wada, Akiko Kukita, Takashi Kakimoto, Ferry Sandra, Kazuko Toh, Kengo Nagata, Tadahiko Iijima, Madoka Horiuchi, Hiromi Matsusaki, Kunio Hieshima, Osamu Yoshie, Hisayuki Nomiyama, RANKL-induced DC-STAMP is essential for osteoclastogenesis., The Journal of experimental medicine, 200, 7, 941-6, 2004.10, Osteoclasts are bone-resorbing, multinucleated giant cells that are essential for bone remodeling and are formed through cell fusion of mononuclear precursor cells. Although receptor activator of nuclear factor-kappaB ligand (RANKL) has been demonstrated to be an important osteoclastogenic cytokine, the cell surface molecules involved in osteoclastogenesis are mostly unknown. Here, we report that the seven-transmembrane receptor-like molecule, dendritic cell-specific transmembrane protein (DC-STAMP) is involved in osteoclastogenesis. Expression of DC-STAMP is rapidly induced in osteoclast precursor cells by RANKL and other osteoclastogenic stimulations. Targeted inhibition of DC-STAMP by small interfering RNAs and specific antibody markedly suppressed the formation of multinucleated osteoclast-like cells. Overexpression of DC-STAMP enhanced osteoclastogenesis in the presence of RANKL. Furthermore, DC-STAMP directly induced the expression of the osteoclast marker tartrate-resistant acid phosphatase. These data demonstrate for the first time that DC-STAMP has an essential role in osteoclastogenesis..
93. Naohisa Wada, Hidefumi Maeda, Yoshito Yoshimine, Akifumi Akamine, Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha., Bone, 10.1016/j.bone.2004.04.023, 35, 3, 629-35, 2004.09, Our recent work showed that human periodontal ligament fibroblasts (HPLF) secrete bioactive osteoprotegerin (OPG), which inhibits osteoclastic differentiation and activity. However, it is unknown how HPLF regulate bone metabolism in the presence of lipopolysaccharide (LPS), which is a cell component of gram-negative bacteria and a pathogen in inflammatory bone diseases such as periodontitis. The present study examined the effects of Escherichia coli LPS on the gene expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), OPG, and receptor activator of NF-kappa B ligand (RANKL) in HPLF using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. In HPLF cultured with LPS for 48 h, expression of both OPG and RANKL mRNA was up-regulated, whereas for up to 24 h of stimulation, such up-regulation was not observed. However, LPS increased expression of IL-1 beta and TNF-alpha mRNA within 6 h of treatment. Moreover, in HPLF cultured with IL-1 beta or TNF-alpha, OPG and RANKL expression was induced within 12 h of culture. The administration of neutralizing antibodies against human IL-1 beta or TNF-alpha to LPS-treated cultures of HPLF inhibited the induction of OPG and RANKL expression. These suggest that LPS stimulates both OPG and RANKL expression in HPLF by up-regulating IL-1 beta and TNF-alpha. In addition, administration of conditioned medium (CM) from HPLF (HPLF-CM) stimulated with LPS for 48 h to mouse bone marrow culture failed to induce osteoclast-like cell (OCL) formation. When mouse spleen cells were cocultured with HPLF in the presence of LPS, OCL formation was completely blocked. Taken together, our results indicate that human periodontal ligament cells stimulated with LPS inhibit osteoclastogenesis by producing more effective OPG than RANKL via the induction of IL-1 beta and TNF-alpha..
94. Kukita T, Wada N, Kukita A, Kakimoto T, Sandra F, Toh K, Nagata K, Iijima T, Horiuchi M, Matsusaki H, Hieshima K, Yoshie O, Nomiyama H., RANKL-induced DC-STAMP is essential for osteoclastogenesis., Journal of Experimental Medicine, 10.1084/jem.20040518, 200, 7, 941-946, vol.200: pp941-946, 2004.06.
95. Wada N, Maeda H, Yoshimine Y, Akamine A, Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF-kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha., Bone, 10.1016/j.bone.2004.04.023, 35, 3, 629-635, vol.35: pp629-635, 2004.04.
96. Hidefumi Maeda, Naohisa Wada, Hiroyoshi Nakamuta, Akifumi Akamine, Human periapical granulation tissue contains osteogenic cells., Cell and tissue research, 10.1007/s00441-003-0832-z, 315, 2, 203-8, 2004.02, Bone defects caused by periapical inflammation can be treated and improved by endodontic therapy. However, the mechanism for osseous healing of periapical lesions after root canal treatment is unclear. In this study we examined whether fibroblastic cells from human periapical granulation tissue could produce calcified matrix in vitro. Periapical lesions from three patients were dissected in endodontic surgery, and fibroblastic cells (HFC) migrating from these lesions in vitro were used in this study. The HFC were cultured with or without beta-glycerophosphate (beta-GP) and ascorbic acid (AA), and the expression of human runt-related transcription factor-2 (Runx2), osterix (Osx), osteopontin (Opn), and osteocalcin (Ocn) mRNA, and alkaline phosphatase (ALPase) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) or by an enzyme-cytochemical technique. Furthermore, calcification in the cells was investigated by von Kossa staining. At the beginning of the culture, HFC expressed Runx2 mRNA faintly, but neither Opn mRNA nor ALPase activity. Immunocytochemical study also showed HFC expressed Runx2 more weakly, compared to SaOS2. However, the expression levels of ALPase, and Runx2, Osx, and Opn mRNA, were stimulated by 2 mM beta-GP and 50 microg/ml AA. After 4 weeks of culture with 2 mM beta-GP and 50 microg/ml AA, HFC formed von Kossa staining-positive calcified deposits on culture dishes, and also expressed Ocn mRNA. These results suggest that inflamed periapical granulation tissue contains osteogenic cells that have the potential to differentiate into mature osteoblastic or cementoblastic cells, and that such cells might contribute to osseous healing after root canal treatment..
97. Watanabe T, Kukita T, Kukita A, Wada N, Toh K, Nagata K, Nomiyama H, Iijima T, Direct stimulation of osteoclastogenesis by MIP-1: evidence obtained from studies using RAW 264 cell clone highly responsive to RANKL., Journal of Endocrinology, 10.1677/joe.0.1800193, 180, 1, 193-201, vol.180: pp193-201, 2004.01.
98. Maeda H, Wada N, Nakamuta H, Akamine A, Human periapical granulation tissue contains osteogenic cells., Cell and Tissue Research, 10.1007/s00441-003-0832-z, 315, 2, 203-208, vol.35: pp203-208, 2004.01.
99. Toshiyuki Watanabe, Toshio Kukita, Akiko Kukita, Naohisa Wada, Kazuko Toh, Kengo Nagata, Hisayuki Nomiyama, Tadahiko Iijima, Direct stimulation of osteoclastogenesis by MIP-1alpha: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL., The Journal of endocrinology, 180, 1, 193-201, 2004.01, Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis..
100. Isamu Hashiguchi, Yoshito Yoshimine, Yasuharu Gotou, Hidefumi Maeda, Naohisa Wada, Akifumi Akamine, Hiroshi Fukuyama, Hidehiko Okumura, An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2002., Fukuoka igaku zasshi = Hukuoka acta medica, 10.15017/18728, 94, 5, 81-6, 2003.05, An epidemiologic examination was carried out to reveal the prevalence of the periodontal diseases and oral pigmentation in patients with Yusho. The results obtained were as follows. 1) 95 patients out of 110 patients, who were examined periodontal pocket depth using Ramfjord' methods, had at least one tooth with periodontal pocket deeper than 3 mm. Similarly, 276 teeth out of a total 495 examined teeth showed periodontal pockets with more than 3 mm depth. However, the ratio of the teeth with periodontal pockets deeper than 4 mm to total examined teeth in each age fell to less than 25%. 2) Oral pigmentation was observed in 75 patients out of 121 patients with Yusho. In this examination, gingival pigmentation was most predominant among oral pigmentation. It is of particular interest that severe pigmentation tended to be observed at a much higher frequency in younger patients with Yusho. Taken these findings into consideration, it was suggested that PCBs and related compounds might play an important role in the development of both periodontal diseases and oral pigmentation..
101. Hashiguchi I, Yoshimine Y, Gotou Y, Maeda H, Wada N, Akamine A, Fukuyama H, Okumura H, An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in yusho patients in 2002. . 94: 81-86, 2003., Fukuoka Acta Medica, vol.94: pp81-86, 2003.01.
102. Wada N, Maeda H, Tanabe K, Tsuda E, Yano K, Nakamuta H, Akamine A, Periodontal ligament cells secrete the factor that inhibits osteoclastic differentiation and function: the factor is osteoprotegerin / osteoclastogenesis inhibitory factor., Journal of Periodontal Research, 10.1034/j.1600-0765.2001.00604.x, 36, 1, 56-63, vol.36: pp56-63, 2001.01.
103. Maeda H, Hashiguchi I, Nakamuta H, Toriya Y, Wada N, Akamine A, Histological study of periapical tissue healing in the Rat Molar after retrofilling with various materials., Journal of Endodontics, 10.1016/S0099-2399(99)80397-5, 25, 1, 38-42, vol.25: pp38-42, 1999.01.