Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Hidefumi Maeda Last modified date:2022.06.23

Professor / Oral Rehabilitation / Department of Dental Science / Faculty of Dental Science

1. Haraguchi A, Yoshida S, Takeshita M, Sumi Y, Mitarai H, Yuda A, Wada H, Nishimura F, Maeda H, Wada N., Effects of ultraviolet irradiation equipment on endodontic disease-related bacteria and dental pulp cells. Effects of ultraviolet irradiation equipment on endodontic disease–related bacteria., Lasers in Dental Science, 2022.03.
2. Yasunaga M, Ishikawa H, Tamaoki S, Maeda H, Ohno J., Embedded human periodontal ligament stem cells spheroids enhance cementogenic differentiation via plasminogen activator inhibitor 1., Int J Mol Sci., 2022.02.
3. Kaneko H, Hasegawa D, Itoyama T, Yoshida S, Tomokiyo A, Hamano S, Sugii H, Maeda H., Inhibition of c-Jun N-terminal kinase signaling promotes osteoblastic differentiation of periodontal ligament stem cells and induces regeneration of periodontal tissues., Arch Oral Biol., 2022.02.
4. Sugii H, Albougha MS, Adachi O, Tomita H, Tomokiyo A, Hamano S, Hasegawa D, Yoshida S, Itoyama T, Maeda H., Activin A promotes osteoblastic differentiation of human pre-osteoblasts through the ALK1-Smad1/5/9 pathway., Int J Mol Sci. , 2021.12.
5. Yoshida S, Sugii H, Itoyama T, Kadowaki M, Hasegawa D, Tomokiyo A, Hamano S, Ipposhi K, Yamashita K, Maeda H., Development of a novel direct dental pulp-capping material using 4-META/MMA-TBB resin with nano hydroxyapatite., Mater Sci Eng C Mater Biol Appl., 10.1016/j.msec.2021.112426, 2021.11.
6. Yamashita K, Tomokiyo A, Ono T, Ipposhi K, Alhasan MA, Tsuchiya A, Hamano S, Sigii H, Yoshida S, Itoyama T, Maeda H., Mineral trioxide aggregate immersed in sodium hypochlorite reduce the osteoblastic differentiation of human periodontal ligament stem cells., Sci Rep, 2021.11, White mineral trioxide aggregate (WMTA) is a root canal treatment material, which is known to exhibit a dark brown color when in contact with sodium hypochlorite solution (NaOCl). This study aimed to investigate the effects of NaOCl on the surface properties of WMTA discs and WMTA-induced osteoblastic differentiation of periodontal ligament stem cells (PDLSCs). Mixed WMTA (ProRoot MTA) was filled into the molds to form WMTA discs. These discs were immersed in distilled water (D-WMTA) or 5% NaOCl (Na-WMTA). Their surface structures and Ca2+ release level was investigated. Moreover, they were cultured with a clonal human PDLSC line (line 1–17 cells). The main crystal structures of Na-WMTA were identical to the structures of D-WMTA. Globular aggregates with polygonal and needle-like crystals were found on D-WMTA and Na-WMTA, which included Ca, Si, Al, C and O. However, many amorphous structures were also identified on Na-WMTA. These structures consisted of
Na and Cl, but did not include Ca. NaOCl immersion also reduced Ca2+ release level from whole WMTA discs. Line 1–17 cells cultured with D-WMTA formed many mineralized nodules and exhibited high expression levels of osteoblast-related genes. However, cells incubated with Na-WMTA generated a small number of nodules and showed low expression levels of osteoblast-related genes. These results indicated that NaOCl reduced Ca2+ release from WMTA by generating amorphous structures and changing its elemental distribution. NaOCl may also partially abolish the ability of WMTA to stimulate osteoblastic differentiation of PDLSCs..
7. Ipposhi K, Tomokiyo A, Ono T, Yamashita K, Alhasan MA, Hasegawa D, Hamano S, Yoshida S, Sugii H, Itoyama T, Ogawa M, Maeda H., Secreted frizzled-related protein 1 promotes odontoblastic differentiation and reparative dentin formation by regulating Notch signaling in dental pulp cells., Cells, 10.3390/cells10092491, 10, 9, 2021.09.
8. Tomokiyo A, Hasegawa D, Ono T, Nagano R, Ipposhi K, Yamashita K, Alhasan MA, Maeda H., Characterization of a clonal human periodontal ligament stem cell line exposed to methacrylate resin-, bioactive glass-, or silicon-based root canal sealers., Odontology, 10.1007/s10266-021-00648-7, 2021.08.
9. Ono T, Tomokiyo A, Ipposhi K, Yamashita K, Alhasan MA, Miyazaki Y, Kunitomi Y, Tsuchiya A, Ishikawa K, Maeda H., Generation of biohybrid implants using a multipotent human periodontal ligament cell line and bioactive core materials., J Cell Physiol, 10.1002/jcp.30336, 2021.06.
10. Maeda H. , Aging and senescence of dental pulp and hard tissues of the tooth., Front Cell Dev Biol., 10.3389/fcell.2020.605996, 2020.11.
11. Maeda H, Mass acquisition of human periodontal ligament stem cells., World J Stem Cells, 10.4252/wjsc.v12.i9.1023, 12, 9, 1023-1031, 2020.09, The periodontal ligament (PDL) is an essential fibrous tissue for tooth retention in the alveolar bone socket. PDL tissue further functions to cushion occlusal force, maintain alveolar bone height, allow orthodontic tooth movement, and connect tooth roots with bone. Severe periodontitis, deep caries, and trauma cause irreversible damage to this tissue, eventually leading to tooth loss through the destruction of tooth retention. Many patients suffer from these diseases worldwide, and its prevalence increases with age. To address this issue, regenerative medicine for damaged PDL tissue as well as the surrounding tissues has been extensively investigated regarding the potential and effectiveness of stem cells, scaffolds, and cytokines as well as their combined applications. In
particular, PDL stem cells (PDLSCs) have been well studied. In this review, I
discuss comprehensive studies on PDLSCs performed in vivo and contemporary
reports focusing on the acquisition of large numbers of PDLSCs for therapeutic
applications because of the very small number of PDLSCs available in vivo..
12. Hasegawa D, Hasegawa K, Kaneko H, Yoshida S, Mitarai H, Arima M, Tomokiyo A, Hamano S, Sugii H, Wada N, Kiyoshima H, Maeda H., MEST Regulates the Stemness of Human Periodontal Ligament Stem Cells., Stem Cells Int., 10.1155/2020/9672673, 2020.07.
13. Yoshida S, Tomokiyo A, Hasegawa D, Hamano S, Sugii H, Maeda H., Insight into the Role of Dental Pulp Stem Cells in Regenerative Therapy., Biology-Basel., 10.3390/biology9070160, 9, 7, 160, 2020.07.
14. Itoyama T, Yoshida S, Tomokiyo A, Hasegawa D, Hamano S, Sugii H, Ono T, Fujino S, Maeda H., Possible function of GDNF and Schwann cells in wound healing of periodontal tissue., J Periodont Res, 10.1111/jre.12774., 2020.06.
15. 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., 2020.04.
16. 藤野翔香、濱野さゆり、糸山知宏、前田英史., 象牙芽細胞分化におけるドーパミンの発現., 日歯内療誌, 10.1002/jcp.29314., 2020.01.
17. Fujino S, Hamano S, Tomokiyo A, Itoyama T, Hasegawa D, Sugii H, Yoshida S, Washio A, Nozu A, Ono T, Wada N, Kitamura C, Maeda H, Expression and function of dopamine in odontoblasts, Journal of Cellular Physiology, 10.1002/jcp.29314, 2019.10.
18. Arima M, Hasegawa D, Yoshida S, Mitarai H, Tomokiyo A, Hamano S, Sugii H, Wada N, Maeda H., R-spondin 2 promotes osteoblastic differentiation of immature human periodontal ligament cells through the Wnt/-catenin signaling pathway., J Periodont Res., 10.1111/jre.12611, 54, 2, 143-153, 2019.02.
19. Toshimitsu T, Kajiya H, Yasunaga M, Maeshiba M, Fujisaki S, Miyaguchi N, Yamaguchi M, Maeda H, Kojima H, Ohno J., Susceptibility of the wnt/Β-catenin pathway accelerates osteogenic differentiation of human periodontal ligament stem cell spheroids., J Hard Tissue Biol, 28(2): 121-128, 2019, org/10.2485/jhtb.28.121, 2019.02.
20. Nozu A, Hamano S, Tomokiyo A, Hasegawa D, Sugii H, Yoshida S, Mitarai H, Taniguchi S, Wada N, Maeda H., Senescence and odontoblastic differentiation of dental pulp cells., J Cell Physiol, 10.1002/jcp.26905, 234, 1, 849-859, ??, 2019.01, 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 odontoblastinductive 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..
21. Yasunaga M, Kajiya H, Toshimitsu T, Nakashima H, Tamaoki S, Ishikawa H, Maeda H, Ohno J., The Early Autophagic Pathway Contributes to Osteogenic Differentiation of Human Periodontal Ligament Stem Cells. , J Hard Tissue Biol, 28(1): 63-70, 2019. , org/10.2485/jhtb.28.63, 28, 1, 2019.01.
22. Daigaku Hasegawa, Naohisa Wada, Shinichiro Yoshida, Hiromi Mitarai, Mai Arima, Atsushi Tomokiyo, Sayuri Hamano, Hideki Sugii, Hidefumi Maeda, Wnt5a suppresses osteoblastic differentiation of human periodontal ligament stem cell-like cells via Ror2/JNK signaling, JOURNAL OF CELLULAR PHYSIOLOGY, 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..
23. Anna Miura, Trang T.H. Tu, Yukiko Shinohara, Lou Mikuzuki, Kaoru Kawasaki, Shiori Sugawara, Takayuki Suga, Takeshi Watanabe, Motoko Watanabe, Yojiro Umezaki, Tatsuya Yoshikawa, Haruhiko Motomura, Miho Takenoshita, Hidefumi Maeda, Akira Toyofuku, Psychiatric comorbidities in patients with Atypical Odontalgia, Journal of Psychosomatic Research, 10.1016/j.jpsychores.2017.11.001, 104, 35-40, 2018.01, Objective Atypical Odontalgia (AO) is a condition characterized by tooth pain with no apparent cause. Although psychiatric comorbidity seems to be very common, it has rarely been studied. To clarify the influence of psychiatric comorbidity on the clinical features in patients with AO, we retrospectively evaluated their examination records. Methods Clinical features and psychiatric diagnoses of 383 patients with AO were investigated by reviewing patients' medical records and referral letters. Psychiatric diagnoses were categorized according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). We also analyzed visual analogue scale (VAS), self-rating depression scale (SDS), and the short-form McGill pain questionnaire (SF-MPQ) scores. Results Of the 383 patients with AO, 177 (46.2%) had comorbid psychiatric disorders. The most common were depressive disorders (15.4%) and anxiety disorders (10.1%). Serious psychotic disorders such as bipolar disorder (3.0%) and schizophrenia (1.8%) were rare. Dental trigger of AO was reported in 217 (56.7%) patients. There were no significant correlations between psychiatric comorbidities and most of the demographic features. Higher VAS and SDS scores, higher frequency of sleep disturbance, and higher ratings of “Fearful” and “Punishing-cruel” descriptors of the SF-MPQ were found in patients with psychiatric comorbidity. Conclusions About half of AO patients had comorbid psychiatric disorders. Dental procedures are not necessarily causative factors of AO. In AO patients with comorbid psychiatric disorders, pain might have a larger emotional component than a sensory one. VAS, SDS, and SF-MPQ scores might aid in the noticing of underlying comorbid psychiatric disorders in AO patients..
24. 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..
25. Hideki Sugii, Alexandre Grimaldi, Jingyuan Li, Carolina Parada, Thach Vu-Ho, Jifan Feng, Junjun Jing, Yuan Yuan, Yuxing Guo, Hidefumi Maeda, Yang Chai, The Dlx5-FGF10 signaling cascade controls cranial neural crest and myoblast interaction during oropharyngeal patterning and development, DEVELOPMENT, 10.1242/dev.155176, 144, 21, 4037-4045, 2017.11, Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here, we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA); however, the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5(-/-) mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays crucial roles in the patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells..
26. 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. (C) 2017 Elsevier Inc. All rights reserved..
27. Serita S, Tomokiyo A, Hasegawa D, Hamano S, Sugii H, Yoshida S, Mizumachi H, Mitarai H, Monnouchi S, Wada N, Maeda H, 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.
28. Hiromi Mitarai, Naohisa Wada, Daigaku Hasegawa, Shinichiro Yoshida, Mai Arima, Atsushi Tomokiyo, Sayuri Hamano, Suguru Serita, Hiroyuki Mizumachi, Hidefumi Maeda, Transgelin mediates TGF-β1-induced proliferation of human periodontal ligament cells., J Peirodont Res, 2017.06.
29. 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-1 beta) 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-1 beta antibody. Compared with untreated cells, HPDLCs treated with IL-1 beta or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1 beta-treated HPDLCs (IL-1 beta-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-1 beta-CM-treated PC12 cells. These stimulatory effects of IL-1 beta-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. (C) 2016 Wiley Periodicals, Inc..
30. S. Monnouchi, H. Maeda, A. Yuda, S. Serita, N. Wada, A. Tomokiyo, A. Akamine, Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells, JOURNAL OF PERIODONTAL RESEARCH, 10.1111/jre.12355, 51, 6, 779-788, 2016.12, Background and Objective: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs.
Material and Methods: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription-polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)-amide (CH-223191), which is an AhR antagonist.
Results: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs.
Conclusions: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking..
31. Shinichiro Yoshida, Naohisa Wada, Daigaku Hasegawa, Miyaji, Hiromi Mitarai, Atsushi Tomokiyo, Sayuri Hamano, Hidefumi Maeda, Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells. , J Dent Res., 10.1177/0022034516653085, 95, 11, 1282-1290, 2016.09, In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability
to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell
activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been
anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated
the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat
dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat
dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation
of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of
the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental
pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at
the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping.
These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A
might be an alternative agent for direct pulp capping, which requires further study..
32. Kyosuke Toyoda, Takao Fukuda, Terukazu Sanui, Urara Tanaka, Kensuke Yamamichi, Ryo Atomura, Hidefumi Maeda, Atsushi Tomokiyo, Takaharu Taketomi, Takeshi Uchiumi, Fusanori Nishimura, Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.25087, 231, 2, 414-427, 2016.02, Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1-17, which possesses the most typical characteristics of PDLSCs. Confocal co-localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1-17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration-associated genes in 1-17 cells. Moreover, Grp78 overexpression enhanced rM180-induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin-induced migration in 1-17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin-induced periodontal tissue regeneration. J. Cell. Physiol. 231: 414-427, 2016. (c) 2015 Wiley Periodicals, Inc..
33. 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, 2647-2660, 2015.11.
34. Urara Tanaka, Terukazu Sanui, Takao Fukuda, Toyoda, K, Taketomi, T, Atomura, R, Yamamichi K, Hidefumi Maeda, Fusanori NISHIMURA, Sprouty2 Inhibition Promotes Proliferation and Migration of Periodontal Ligament Cells., Oral Dis., 10.1111/odi.12369, 21, 8, 977-986, 2015.11, We previously demonstrated that a dominant-negative Sprouty2 (Spry2) mutation promotes osteoblast proliferation and differentiation after basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulation, whereas it diminishes proliferation of gingival epithelial cells, thereby inducing favourable conditions for periodontal tissue regeneration. In this study, we investigated how Spry2 inhibition affects the cellular physiology of periodontal ligament (PDL) cells.
A total of 1-17 PDL cells (multipotent clonal human PDL cell line) were stimulated with bFGF and EGF after transfection of Spry2 siRNA. Cell proliferation, migration, ALP staining, real-time PCR, Western blot and immunofluorescence assays were performed.
ERK1/2 activation and proliferation of 1-17 PDL cells were significantly upregulated by the addition of Spry2 siRNA in the presence of bFGF and EGF. In addition, Spry2 siRNA reduced transcription of osteogenesis-related genes and ALP staining relative to control cells. Furthermore, it increased AKT/phosphatidylinositol 3-kinase (PI3K) phosphorylation; consequently, Rac1 but not Cdc42 was activated, thereby promoting lamellipodia formation, cell proliferation and migration after stimulation by bFGF and EGF.
Spry2 combined with bFGF and EGF stimulation reduced PDL cell migration and proliferation with inducing osteoblastic differentiation. These in vitro findings may provide a molecular basis for novel therapeutic approaches for establishing periodontal tissue regeneration..
35. Myna N. Zakaria, 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., 10.1111/iej.12361, 48, 8, 717-728, 2015.08, AIM:

To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach.

Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers.

Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples.

Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species..
36. S. Monnouchi, H. Maeda, A. Yuda, S. Hamano, N. Wada, A. Tomokiyo, K. Koori, H. Sugii, S. Serita, A. Akamine, Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells, JOURNAL OF PERIODONTAL RESEARCH, 10.1111/jre.12200, 50, 2, 231-239, 2015.04, Background and ObjectiveThe periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17).
Material and MethodsHuman primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining.
ResultsPositive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2.
ConclusionMechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11..
37. Ayako Washio, Aika Nakagawa, Tatsuji Nishihara, Hidefumi Maeda, Chiaki Kitamura, Physicochemical properties of newly developed bioactive glass cement and its effects on various cells, JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS, 10.1002/jbm.b.33209, 103, 2, 373-380, 2015.02, Biomaterials used in dental treatments are expected to have favorable properties such as biocompatibility and an ability to induce tissue formation in dental pulp and periapical tissue, as well as sealing to block external stimuli. Bioactive glasses have been applied in bone engineering, but rarely applied in the field of dentistry. In the present study, bioactive glass cement for dental treatment was developed, and then its physicochemical properties and effects on cell responses were analyzed. To clarify the physicochemical attributes of the cement, field emission scanning electron microscopy, X-ray diffraction, and pH measurement were carried out. Cell attachment, morphology, and viability to the cement were also examined to clarify the effects of the cement on odontoblast-like cells (KN-3 cells), osteoblastic cells (MC3T3-E1 cells), human periodontal ligament stem/progenitor cells and neuro-differentiative cells (PC-12 cells). Hydroxyapatite-like precipitation was formed on the surface of the hardened cement and the pH level changed from pH10 to pH9, then stabilized in simulate body fluid. The cement had no cytotxic effects on these cells, and particulary induced process elongation of PC-12 cells. Our results suggest that the newly developed bioactive glass cement have capability of the application in dental procedures as bioactive cement. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 373-380, 2015..
38. Asuka Yuda, Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohide Yamamoto, Naohisa Wada, Katsuaki Koori, Atsushi Tomokiyo, Sayuri Hamano, Daigaku Hasegawa, Akifumi Akamine, Effect of CTGF/CCN2 on Osteo/Cementoblastic and Fibroblastic Differentiation of a Human Periodontal Ligament Stem/Progenitor Cell Line, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.24693, 230, 1, 150-159, in press, 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-1 (TGF-1) significantly promoted type I collagen and fibronectin expression compared with that of TGF-1 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. J. Cell. Physiol. 229: 150-159, 2014. (c) 2014 Wiley Periodicals, Inc..
39. 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, 1249-1262, 2014.10, The population of the world grows every year, and life expectancy tends to increase. Thus, long term preservation of teeth in aged individuals is an urgent issue. The main causes of tooth loss are well known to be periodontitis, caries, fractures, and orthodontic conditions. Although implant placement is a widely accepted treatment for tooth loss, most patients desire to preserve their own teeth. Many clinicians and researchers are therefore challenged to treat and preserve teeth that are irreversibly affected by deep caries, periodontitis, fractures, and trauma. Tissue engineering techniques are beneficial in addressing this issue; stem cells, signal molecules, and scaffolds are the main elements of such techniques. In this review, we describe these three elements with respect to their validation for regeneration of the periodontium and focus particularly on the potency of diverse scaffolds. In addition, we provide a short overview of the ongoing studies of 4 methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl-borane resin including calcium or hydroxyapatite for periodontium regeneration..
40. Hidefumi Maeda, Akifumi Akamine, Quest for the development of tooth root/periodontal ligament complex by tissue engineering., Integr Mol Med., 10.15761/IMM.1000106, 1, 2, 22-25, 2014.10, The life-span of the tooth is intimately-associated with healthiness of periodontal ligament (PDL) which is a connective tissue situated between bone and cementum
that covers tooth root surface. However, once this tissue is severely damaged by deep caries, periodontitis, and trauma, this leads to severe difficulty in its regeneration,
resulting in tooth loss and decreased quality of life. The development of the therapy for generation and regeneration of the periodontal tissue is an urgent issue.
Therefore, researchers have tried to improve efficiently-generative and regenerative medicine using stem cells, signal molecules, and scaffolds, requisite for tissue
regeneration. In recent studies, a dental follicle tissue that is composed of stem cell population potentially differentiating into PDL tissue, cementum, and alveolar
bone is of current interest. More recently a revolutionary and attractive study reporting the development of bio-hybrid implant that reserved newly-formed cementum/
PDL tissue complex on its surface was introduced. In this review, we describe comprehensive reports that tried to develop the cementum/PDL complex by tissue
engineering and future prospects..
41. Naohisa Wada, Hidefumi Maeda, Daigaku Hasegawa, Stan Gronthos, Peter Mark Bartold, Danijela Menicanin, Shinsuke Fujii, Shinichiro Yoshida, Atsushi Tomokiyo, Satoshi Monnouchi, Akifumi Akamine, Semaphorin 3A Induces Mesenchymal-Stem-Like Properties in Human Periodontal Ligament Cells, STEM CELLS AND DEVELOPMENT, 10.1089/scd.2013.0405, 23, 18, 2225-2236, 2014.09, 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..
42. Katsuaki Koori, Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, Giichiro Kawachi, Daigaku Hasegawa, Sayuri Hamano, Hideki Sugii, Naohisa Wada, Akifumi Akamine, The roles of calcium-sensing receptor and calcium channel in osteogenic differentiation of undifferentiated periodontal ligament cells, CELL AND TISSUE RESEARCH, 10.1007/s00441-014-1918-5, 357, 3, 707-718, 2014.09, 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..
43. Yoko Teramatsu, Hidefumi Maeda, Hideki Sugii, Atsushi Tomokiyo, Sayuri Hamano, Naohisa Wada, Asuka Yuda, Naohide Yamamoto, Katsuaki Koori, Akifumi Akamine, Expression and effects of epidermal growth factor on human periodontal ligament cells, CELL AND TISSUE RESEARCH, 10.1007/s00441-014-1877-x, 357, 3, 633-643, 2014.09, 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-1 beta were found to be upregulated in a perilesional pattern. Interleukin-1 beta induced EGF expression in HPDLCs but not EGFR. It also increased transforming growth factor-alpha (TGF-alpha) 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..
44. 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-beta (TGF-beta) superfamily and a dimer of inhibini beta a, 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-1 beta expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1 beta or TNF-alpha also up-regulated the expression of the gene encoding inhibin beta a.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. (C) 2014 Elsevier Inc. All rights reserved..
45. Takashi Tsutsumi, Hiroshi Kajiya, Teruhisa Fukawa, Mina Sasaki, Tetsuomi Nemoto, Takashi Tsuzuki, Yutaka Takahashi, Shinsuke Fujii, Hidefumi Maeda, Koji Okabe, The potential role of transient receptor potential type A1 as a mechanoreceptor in human periodontal ligament cells, European Journal of Oral Sciences, 10.1111/eos.12083, 121, 6, 538-544, 2013.12, Transient receptor potential type A1 (TRPA1) is reported to be a Ca2+-permeable channel and is activated by cold temperatures and mechanical stimuli in the hair cells and in dorsal root ganglion. Using a DNA microarray, we found that TRPA1 was significantly up-regulated in human periodontal ligament (hPDL) cells 2 d after intermittent mechanical stimulation (iMS) loading compared with unloaded cells. Although hPDL cells are known to respond to mechanical stimulation induced by occlusal force, little is known about the expression and functional role of TRPA1 in these cells. Therefore, we investigated the effects of iMS on TRPA1 expression and its signaling pathway in hPDL cells. Intermittent mechanical stimulation loading up-regulated TRPA1 expression in hPDL cells in a time-dependent manner, but had no effect on other mechanoreceptors. Furthermore, iMS significantly increased the phosphorylation of mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, and the expression of C-C chemokine ligand 2 (CCL2). Transient receptor potential type A1 agonists also increased MAPK phosphorylation and the intracellular Ca2+ concentration. By contrast, inhibition or silencing of TRPA1 partially suppressed iMS-induced MAPK phosphorylation. In summary, iMS during occlusion activates TRPA1 and MAPK signaling in periodontal ligament tissues, suggesting that TRPA1 regulates the mechanosensitivity of occlusal force via activation of MAPKs in hPDL cells. © 2013 Eur J Oral Sci..
46. Hidefumi Maeda, Shinsuke Fujii, Atsushi Tomokiyo, Naohisa Wada, Akifumi Akamine, Periodontal Tissue Engineering: Defining the Triad, INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS, 10.11607/jomi.te26, 28, 6, E461-E471, 2013.11, The idea that somatic stem cells are localized in periodontal ligament (PDL) tissues as PDL stem cells (PDLSCs) responsible for construction and reconstruction of the periodontium has been widely accepted. Many dental scientists have attempted to clarify the identity of these PDLSCs, but the number of PDLSCs localized in PDL tissues is too small to be routinely and conveniently analyzed. Therefore, researchers have been attempting to develop undifferentiated PDL cell lines by transducing them with genes that are suitable for immortalization. The present authors were the first to succeed in establishing two clonal human PDL stem/progenitor cell lines that possessed multipotency derived from PDL tissues and that expressed PDL-related molecules as well as neural crest-and embryonic stem-related markers. The differentiation stages of these cell lines appeared to vary based on their potential to differentiate into other lineage cells, their response to tissue regeneration-related cytokines, and their behavior when transplanted into immunodeficient rats. This review describes the phenotypes of these cell lines compared with reported PDLSCs or other MSCs and discusses contemporary circumstances related to PDL regenerative medicine. Differential analyses between these two clones will reveal the mechanism of differentiation of PDLSCs as well as their phenotypes. The results will also allow for the acquisition of a mass population of PDLSCs or other stem cells directed toward PDL-lineage cells and to develop an unmet treatment needed for construction and reconstruction of PDL tissues based on tissue engineering techniques..
47. 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, 249-263, 2013.05.
48. 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, 3093-3103, 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 (a-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. J. Cell. Biochem. 113: 30933103, 2012. (c) 2012 Wiley Periodicals, Inc..
49. 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, 556-564, 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-1 beta), or tumour necrosis factor-alpha (TNF-a) 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-1 beta and TNF-a 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 aV beta 3 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 aV beta 3. These findings suggest that GDNF may contribute to wound healing in PDL tissue..
50. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohisa Wada, Kiyomi Kono, Naohide Yamamoto, Katsuaki Koori, Yoko Teramatsu, Akifumi Akamine, A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.22933, 227, 5, 2040-2050, 2012.05, Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration. J. Cell. Physiol. 227: 2040-2050, 2012. (C) 2011 Wiley Periodicals, Inc..
51. Hidefumi Maeda, Shinsuke Fujii, Satoshi Monnouchi, Naohisa Wada, Akifumi Akamine, Differentiation of periodontal ligament stem/progenitor cells: Roles of TGF-β1, Stem Cells and Cancer Stem Cells, Volume 4: Therapeutic Applications in Disease and Injury, 10.1007/978-94-007-2828-8_5, 51-58, 2012.01, Tooth loss provokes loss of occlusal function, resulting in a decreased quality of life. Tooth loss is related primarily to irretrievable damage of the periodontium caused by deep caries, severe periodontal diseases, or irreversible trauma. Periodontal ligament (PDL) is a central tissue in periodontium and contains stem/progenitor cells that are believed to have the potential to regenerate periodontium, including PDL tissue. For this reason, we and other groups have focused on such undifferentiated cells to elucidate their applicable potentials for PDL regenerative medicine based on cell-based tissue engineering. However, how their differentiation is controlled remains unclear. We recently suggested that transforming growth factor-beta 1 is a candidate factor for regulation of fibroblastic differentiation of stem/progenitor cells in PDL tissue..
52. 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, 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..
53. 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.
54. Biocompatibility of resin-based sealers- Comparison of Superbond sealer with AH Plus -.
55. Kwon SM, Kim SA, Fujii S, Maeda H, Ahn SG, Yoon JH, Transforming growth factor β1 promotes migration of human periodontal ligament cells through heat shock protein 27 phosphorylation., Biological & pharmaceutical bulletin, 10.1248/bpb.34.486, 34, 4, 486-489, 2011.04.
56. [An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2010]..
57. S. Monnouchi, H. Maeda, S. Fujii, A. Tomokiyo, K. Kono, A. Akamine, The Roles of Angiotensin II in Stretched Periodontal Ligament Cells, JOURNAL OF DENTAL RESEARCH, 10.1177/0022034510382118, 90, 2, 181-185, 2011.02, The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-beta 1 (TGF-beta 1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-beta 1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-beta 1 and ALP..
58. 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, 233-242, 2010.11.
59. Hidefumi Maeda, Tsuguhisa Nakano, Atsushi Tomokiyo, Shinsuke Fujii, Naohisa Wada, Satoshi Monnouchi, Kiyomi Hori, Akifumi Akamine, Mineral Trioxide Aggregate Induces Bone Morphogenetic Protein-2 Expression and Calcification in Human Periodontal Ligament Cells, JOURNAL OF ENDODONTICS, 10.1016/j.joen.2009.12.024, 36, 4, 647-652, 2010.04, 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. (J Endod 2010;36:647-652).
60. Yoshiyuki Yasuda, Yuki Tatematsu, Shinsuke Fujii, Hidefumi Maeda, Akifumi Akamine, Mahmoud Torabinejad, Takashi Saito, Effect of MTAD on the Differentiation of Osteoblast-like Cells, JOURNAL OF ENDODONTICS, 10.1016/j.joen.2009.11.002, 36, 2, 260-263, 2010.02, Introduction: The aim of the present study was to investigate the effect of MTAD on the differentiation of osteoblast-like cells. Methods: The cell viability assay was performed to evaluate the cytotoxicity of MTAD on MC3T3-E1 and periodontal ligament cells at the various concentrations. The bone sialoprotein (BSP) gene expression was also examined by real-time polymerase chain reaction. Results: MTAD exhibited a lower cytotoxicity compared with other intracanal irrigants and medication. The MC3T3-E1 cells treated with H(2)O(2) showed a decrease in the alkaline phosphatase (ALP) activity by 40% on day 14 compared with the control group at the concentration of 50 mu g/mIL. No significant difference in the ALP activity was observed between MTAD and control group. Furthermore, MTAD and Ca(OH)(2) paste did not change in BSP gene expression in MC3T3-E1 cells on day 21. Conclusions: These results suggested that MTAD is a less cytotoxic irrigant and does not affect differentiation into osteoblasts compared with other intracanal irrigants, such as H(2)O(2), NaOl, ethylenediaminetetraacetic acid, and chlorhexidine. (J Endod 2010;36:260-263).
61. The Effects of MTA on Human Periodontal Ligament Cells..
62. An Epidemiologic Examination on the Prevalence of the Periodontal Diseases and Oral Pigmentation in Yusho Patients in 2008..
63. [An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2008]..
64. Y. Yasuda, H. Inuyama, H. Maeda, A. Akamine, J. E. Noer, T. Saito, Cytotoxicity of one-step dentin-bonding agents toward dental pulp and odontoblast-like cells, JOURNAL OF ORAL REHABILITATION, 10.1111/j.1365-2842.2008.01885.x, 35, 12, 940-946, 35(12):940-6, 2008, 2008.12, The purpose of this study was to compare the cytotoxicity of five one-step dentin-bonding agents on human dental pulp and odontoblast-like cells (MDPC-23). Photopolymerized and unpolymerized samples of these dentin-bonding agents were prepared and incubated with dental pulp or MDPC-23 cells. After 24 or 72 h of incubation, the number of unstained cells with trypan blue was counted. The staining of cells with trypan blue stands for a cytotoxicity. The pulp cell and MDPC-23 cytotoxicity of polymerized sample treatment increased in the order of AQ Bond Plus (AQ)< Clearfil Tri-S Bond (TS)=G-bond (GB)< Absolute (AB)< Adper Prompt (AP) for 24 and 72 h. The pulp cell cytotoxicity of unpolymerized sample treatment for 24 h increased in the order of AQ < GB = AB < TS < AP. The MDPC-23 cytotoxicity of unpolymerized sample treatment for 24 h increased in the order of AQ < GB < TS = AB < AP. Whether polymerized or unpolymerized, AQ was the least cytotoxic agent, while AP was the strongest. All polymerized dentin-bonding agents exhibited lower cytotoxicity by 2-65% than their unpolymerized counterparts. The appearance of the cytotoxicity of dentin-bonding agents was time-dependent, and cell viability was lower at 72 h by 2-46% than at 24 h. The cytotoxicity to MDPC-23 cells was about 5-24% higher than that to pulp cells. These results indicate that one-step dentin-bonding agents differ markedly in their cytotoxicity. Differential cytotoxic effects of one-step dentin-bonding agents should be considered during clinical application of operative restoration..
65. Yoshio Kano, Noboru Horie, Shima Doi, Fumika Aramaki, Hidefumi Maeda, Fukumi Hiragami, Kenji Kawamura, Hirotoshi Motoda, Yoshihisa Koike, Junichi Akiyama, Sueo Eguchi, Ken Hashimoto, Artepillin C derived from propolis induces neurite outgrowth in PC12m3 cells via ERK and p38 MAPK pathways, NEUROCHEMICAL RESEARCH, 10.1007/s11064-008-9633-9, 33, 9, 1795-1803, 33(9):1795-803 2008, 2008.09, We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 mu M, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells..
66. Shinsuke Fujii, Hidefumi Maeda, Naohisa Wada, Atsushi Tomokiyo, Masahiro Saito, Akifumi Akamine, Investigating a clonal periodontal ligament progenitor/stem cell line in vitro human and in vivo, JOURNAL OF CELLULAR PHYSIOLOGY, 10.1002/jcp.21359, 215, 3, 743-749, 215(3):743-749, 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 CID 146 was expressed in cell lines 1-4 and I - I I and that STRC-I was expressed in lines 1-11 and 1-24. Lines 1-4 and I - I I 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 P-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..
67. Atsushi Tomokiyo, Hidefumi Maeda, Shinsuke Fujii, Naohisa Wada, Kazuya Shima, Akifumi Akamine, Development of a multipotent clonal human periodontal ligament cell line, DIFFERENTIATION, 10.1111/j.1432-0436.2007.00233.x, 76, 4, 337-347, 76(4):337-347, 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..
68. SEM images of root canal dentin irrigated with EDTA and NaOCl - Comparison with ultrasonic irrigation -.
69. Cytotoxicity of one-step adhesive resins on dental pulp cells and their effects on differentiation to odontoblasts..
70. An Epidemiological Examination on the Prevalence of the Periodontal Diseases and Oral Pigmentation in Yusho Patients in 2006..
71. [Epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2006]..
72. In vitro evaluation of cytotoxicity of resin-based root canal sealer.
73. Ganwei Lu, Hidefumi Maeda, Sakamuri V. Reddy, Noriyoshi Kurihara, Robin Leach, Judith L. Anderson, G. David Roodman, Cloning and characterization of the annexin II receptor on human marrow stromal cells, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M607072200, 281, 41, 30542-30550, 281(41): 30542-30550, 2006.10, Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. I-125-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a K-d of 5.79 nM and B-max of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using I-125-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation..
74. Effects of root-end filling materials on the osteoblast-like differentiation of human periodontal ligament cells.
75. S Fujii, H Maeda, N Wada, Y Kano, A 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, 117-125, 324(1): 117-125, 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 mu 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..
76. H Maeda, N Wada, S Fujii, A 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, 135-140, 324(1): 117-125, 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 β-glycerophosphate (P-GP) and 50 μ g/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 β-GP and 50 μ g/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 Xray 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..
77. N Wada, H Maeda, Y Yoshimine, A 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, 629-635, 35 (3) 629-635., 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- I beta (IL-1beta), 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-1beta and TNF-alpha mRNA within 6 h of treatment. Moreover, in HPLF cultured with IL- 1beta or TNF-alpha., OPG and RANKL expression was induced within 12 h of culture. The administration of neutralizing antibodies against human IL-1beta 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- 1beta 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-1beta and TNF-alpha. (C) 2004 Elsevier Inc. All rights reserved..
78. H Maeda, N Wada, H Nakamuta, A Akamine, Human periapical granulation tissue contains osteogenic cells, CELL AND TISSUE RESEARCH, 10.1007/s00441-003-0832-z, 315, 2, 203-208, 315 (2): 203-208, 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 mug/ml AA. After 4 weeks of culture with 2 mM beta-GP and 50 mug/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..
79. An epidemiologic examination on the prevalence of the periodontal diseases and oral pigmentation in Yusho patients in 2002..
80. M Koide, H Maeda, JL Roccisana, N Kawanabe, SV Reddy, Cytokine regulation and the signaling mechanism of osteoclast inhibitory peptide-1 (OIP-1/hSca) to inhibit osteoclast formation, JOURNAL OF BONE AND MINERAL RESEARCH, 10.1359/jbmr.2003.18.3.458, 18, 3, 458-465, 18 (3): 458-465, 2003.03, The osteoclast (OCL) is the primary bone resorbing cell. OCL formation and activity is regulated by local factors produced in the bone microenvironment. We recently identified OCL inhibitory peptide-1 (OIP-1/hSca) as a novel inhibitor of OCL formation and bone resorption that is produced by OCLs. OIP-1 is a glycosylphosphatidyl-inositol (GPI)-linked membrane protein (16 kDa) related to the mouse Ly-6 family of hematopoietic proteins. OIP-1 mRNA is expressed in human OCL precursors, granulocyte-macrophage colony-forming unit (GM-CFU), bone marrow cells, and osteoblast cells. We used cycle-dependent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, which further demonstrated that interferon-gamma (IFN-gamma) strongly enhanced OIP-1/hSca mRNA expression in bone marrow cells and GM-CFU. Similarly, interleukin (IL)-1beta also enhanced OIP-1 mRNA expression in GM-CFU. To determine the participation of OIP-1 in IFN-gamma inhibition of OCL formation, we tested the capacity of a neutralizing antibody specific to OIP-1 c-peptide to inhibit IFN-gamma's effects on OCL-like cell differentiation of mouse macrophages, RAW 264.7 cells. Anti-OIP-1 c-peptide specific antibody partially neutralized IFN-gamma inhibition of OCL differentiation. Furthermore, OIP-1 inhibited phospho-c-Jun (p-c-jun) kinase activity in RAW 264.7 cells. However, OIP-1/hSca did not affect NF-kappaB activation in these cells. Western blot analysis further demonstrated that OIP-1 significantly decreased TNF receptor associated factor 2 (TRAF-2) expression in RAW 264.7 cells. However, OIP-1 had no effect on TRAF-6 expression in these cells. These data show that IFN-gamma enhances OIP-1/hSca expression in OCL precursors, GM-CFU, and that OIP-1 inhibits OCL formation through suppression of TRAF-2 and p-c-Jun kinase activity..
81. M Koide, N Kurihara, H Maeda, SV Reddy, Identification of the functional domain of osteoclast inhibitory peptide-1/hSca, JOURNAL OF BONE AND MINERAL RESEARCH, 17, 1, 111-118, 17 (1): 111-118, 2002.01, Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-1/human Sea [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16 kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1 c-peptide (10-100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] induced OCL formation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore, the c-peptide (10-100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1 and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment..
82. Goto T, Maeda H, Tanaka T., A selective inhibitor of matrix metalloproteases inhibits the migration of isolated osteoclasts by increasing the life span of podosomes., Journal of Bone and Mineral Metabolism, 10.1007/s007740200013, 20, 2, 98-105, 20 (2): 98-105, 2002.01.
83. N Kurihara, C Menaa, H Maeda, DJ Haile, SV Reddy, Osteoclast-stimulating factor interacts with the spinal muscular atrophy gene product to stimulate osteoclast formation, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M100233200, 276, 44, 41035-41039, 276 (44): 41035-41039, 2001.11, We have recently identified and cloned an intracellular peptide termed osteoclast-stimulating factor (OSF) that increases osteoclast (OCL) formation and bone resorption through a cellular signal transduction cascade, possibly through its interaction with c-Src or related family members. To further identify participants in the OSF signaling cascade, we used yeast two-hybrid screening with Saccharomyces cerevisiae, and we found that the 40-kDa spinal muscular atrophy disease-determining gene product, survival motor neuron (SMN), interacts with the OSF-Src homology 3 domain. Reverse transcription-polymerase chain reaction analysis of SMN mRNA expression in cells of the OCL lineage demonstrates that expression of the exon 7 splice variant of SMN is restricted to mature OCLs, whereas the unspliced transcript was expressed in OCL precursors as well as mature OCLs. Treatment of murine bone marrow cultures with conditioned media (5% (v/v)) from 293 cells transiently expressing the SMN cDNA significantly increased OCL formation, compared with treatment with conditioned media from mock-transfected cells. Furthermore, OCL-stimulatory activity by OSF or SMN was abolished by antisense constructs to SMN or OSF, respectively. These data confirm the participation of SMN in the OSF-enhanced expression of an OCL stimulator. OSF-SMN interaction may provide more insights into novel cellular signaling mechanisms that may play an important role in congenital bone fractures associated with type I spinal muscular atrophy disease..
84. N Wada, H Maeda, K Tanabe, E Tsuda, K Yano, H Nskamuta, A Akamine, Periodontal ligament cells secrete the factor that inhibits osteoclastic differentiation and function: the factor is ostroprotegerin/osteoclastogenesis inhibitory factor, JOURNAL OF PERIODONTAL RESEARCH, 36, 1, 56-63, 36 (1): 56-63, 2001.02, The periodontal ligament, a highly specialized connective tissue situated between the tooth and the alveolar bone of the tooth socket, has been thought to influence the remodeling of the alveolar bone. The effects of two human periodontal ligament fibroblastic cell populations (HPLFs) on osteoclast-like cell (OCL) formation and the function of authentic osteoclasts were examined. The addition of the conditioned media (CM) from both HPLF cultures (HPLF-CMs) to mouse bone marrow culture inhibited OCL formation in spite of the presence of 10 (8) M 1 alpha, 25 dihydroxyvitamin D-3 (1 alpha ,25(OH)(2)D-3). This inhibitory effect was most remarkable when both CMs were added during day 6 to day 9 following bone marrow culture, just at the late stage of OCL differentiation. HPLF-CMs also induced a significant decrease in the pit area and the pit number formed by authentic osteoclasts on ivory slices. The administration of neutralizing monoclonal antibody (OI-l) against human osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) with (HPLF-CMs) to mo use bone marrow culture almost completely blocked the inhibitory effect of these CMs on OCL formation. Immunofluorescent examination of HPLF with OI-l revealed intense positive reactivity in the cytoplasm. Western blot analysis of HPLF-CM using anti-human OPG/OCIP polyclonal antibody resulted in the detection of bands of 60 kDa and 120 kDa which were consistent with those of OPG/OCIF. These results suggest that HPLF cells product and secrete OPG;OCIF, and that this factor from HPLF prevents the differentiation of the late preosteoclast and the function of the mature osteoclasts..
85. C Menaa, SV Reddy, N Kurihata, H Maeda, D Anderson, T Cundy, J Cornish, FR Singer, JM Bruder, GD Roodman, Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget's disease of bone, JOURNAL OF CLINICAL INVESTIGATION, 105, 12, 1833-1838, 105 (12): 1833-1838, 2000.06, Paget's disease is characterized by highly localized areas of increased osteoclast (OCL) activity. This suggests that the microenvironment in pagetic lesions is highly osteoclastogenic, or that OCL precursors in these lesions are hyperresponsive to osteoclastogenic factors (or both). To examine these possibilities, me compared RANK ligand (RANKL) mRNA expression in a marrow stromal cell line developed from a pagetic lesion (PSV10) with that in a normal stromal cell line (Saka), and expression in marrow samples from affected bones of Paget's patients with that in normal marrow. RANKL mRNA was increased in PSV10 cells and pagetic marrow compared with Saka cells and normal marrow, and was also increased in marrow from affected bones compared with uninvolved bones from Paget's patients. Furthermore, pagetic marrow cells formed OCLs at much lower RANKL, concentrations than did normal marrow. Anti-IL-6 decreased the RANKL responsivity of pagetic marrow to normal levels, whereas addition of IL-6 to normal marrow enhanced RANKL responsivity. Thus, RANKL expression and responsivity is increased in pagetic lesions, in part mediated by IL-6. These data suggest that the combination of enhanced expression of RANKL in affected bones and increased RANKL sensitivity of pagetic OCL precursors may contribute to the elevated numbers of OCLs in Paget's disease..
86. K Tanabe, H Nakanish, H Maeda, T Nishioku, K Hashimoto, SY Liou, A Akamine, K Yamamoto, A predominant apoptotic death pathway of neuronal PC12 cells induced by activated microglia is displaced by a non-apoptotic death pathway following blockage of caspase-3-dependent cascade, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.274.22.15725, 274, 22, 15725-15731, 274 (22): 15725-15731, 1999.05, Activated microglia have been implicated in the regulation of neuronal cell death, However, the biochemical mechanism for neuronal death triggered by activated microglia is still unclear, When treated with activated microglia, neuronal PC12 cells undergo apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation, Apoptotic bodies formed were subsequently phagocytosed by neighboring activated microglia. Pretreatment of the cells with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death. Although Bcl-2 overexpression in the cells caused the inhibition of caspase-3-like protease activity and DNA fragmentation and the effective interference of apoptosis induced by deprivation of trophic factors, it could not suppress the activated microglia-induced neuronal death, At the electron microscopic level, degenerating cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular-shaped masses, severely disintegrated perikarya, and marked vacuolation. Various protease inhibitors tested did not inhibit this cell death, whereas the radical oxygen species scavenger N-acetyl-L-cysteine significantly suppressed this death. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade..
87. H Maeda, K Akasaki, Y Yoshimine, A Akamine, K Yamamoto, Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts, HISTOCHEMISTRY AND CELL BIOLOGY, 111, 4, 245-251, 1999.04, Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post-and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts..
88. H Maeda, Hashiguchi, I, H Nakamuta, Y Toriya, N Wada, A Akamine, Histological study of periapical tissue healing in the rat molar after retrofilling with various materials, JOURNAL OF ENDODONTICS, 25, 1, 38-42, 25 (1): 38-42, 1999.01, We histologically examined the effects on the periapical tissue of various dental filling materials applied as retrofillings in rats and compared them with those of amalgam. The 4-META-TBB resin Superbond and the light-cured composite resin produced the least severe inflammatory reaction, with the greatest amount of new bone. In these specimens, regeneration of a part of the periodontal ligament was also observed. These results indicate that these materials might be very biocompatible and thus foster the natural regeneration of the periapical tissue..
89. Maeda H, Akasaki K, Yoshimine Y, Akamine A, and Yamamoto K., Limited and Selective Localization of the Lysosomal Membrane Glycoproteins LGP85 and LGP96 in Rat Osteoclasts., Histochemistry and Cell Biology, 10.1007/s004180050354, 111, 4, 245-251, 111 (4): 245-251, 1999.01.
90. Kukita A, Kukita T, Ouchida M, Maeda H, Yatsuki H, and Kohashi O., Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional repressor, is involved in osteoclastogenesis., Blood, 94, 6, 1987-1997, 94 (6): 1987-1997, 1999.01.
91. T Kukita, A Kukita, L Xu, H Maeda, T Iijima, Successful detection of active osteoclasts in situ by systemic administration of an osteoclast-specific monoclonal antibody, CALCIFIED TISSUE INTERNATIONAL, 10.1007/s002239900506, 63, 2, 148-153, 63 (2): 148-153, 1998.08, Cell-surface proteins preferentially expressed on osteoclasts are thought to play important roles in the functional modulation of the osteoclasts. Recently, we found a novel cell-surface antigen designated Kat1-antigen (Kat1-Ag) specifically expressed on rat osteoclasts. It would be useful to regulate the functional activity of the osteoclasts directly via an osteoclast-specific antigen expressed on the cell surface of the osteoclasts. In order to establish the basis of such an application, in the present study we established a method for the direct detection of osteoclasts in situ by a systemic administration of the anti-Kat1-Ag monoclonal antibody (mAb Kat1) to rats, and we successfully detected functional osteoclasts in situ. Prior to performing in vivo experiments, we examined the reactivity of the mAb Kat1 to the isolated rat osteoclasts. Approximately 40-80% of the osteoclasts were reactive with mAb Kat1, suggesting that this mAb recognizes osteoclasts in a specific differentiation or functional state. Calcitonin treatment of osteoclast-like cells formed in vitro from bone marrow cells resulted in a conversion of Kat1-positive osteoclast-like cells into Kat1-negative multinucleated cells, showing the positive correlation between the Kat1-Ag expression and the potential bone-resorbing activity of osteoclasts, Administration of this lineage-specific mAb to the peritoneal cavity of newborn rats resulted in a successful recruitment of mAb Kat1 to the newly formed osteoclasts and functional osteoclasts in a highly specific manner. Detailed analysis by immunoelectron microscopy revealed that this mAb specifically bound to the basolateral side of the active osteoclasts, which were identified by their typical ruffled border and clear zone, whereas the mAb did not react to postfunctional osteoclasts. These findings demonstrate a high potential utility of mAb Kat1 in osteoclast-targeted regulation of bone remodeling..
92. Tsukuba T, Sakai H, Yamada M, Maeda H, Hori H, Azuma T, Akamine A, and Yamamoto K., Biochemical Properties of the Monomeric Mutant of Human Cathepsin E Expressed in Chinese Hamster Ovary Cells: Comparison with Dimeric Forms of the Natural and Recombinant Cathepsin E., J Biochemistry, 119, 1, 126-134, 119 (1): 126-134, 1996.01.
93. T Tsukuba, H Sakai, M Yamada, H Maeda, H Hori, T Azuma, A Akamine, K Yamamoto, Biochemical properties of the monomeric mutant of human cathepsin E expressed in Chinese hamster ovary cells: Comparison with dimeric forms of the natural and recombinant cathepsin E, JOURNAL OF BIOCHEMISTRY, 10.1093/oxfordjournals.jbchem.a021197, 119, 1, 126-134, 1996.01, Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys(43) in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys(43)-->Ser(43)) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH7 and 37 degrees C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo..
94. Kukita T, Kukita A, Nagata K, Maeda H, Kurisu K, Watanabe T, and Iijima T, Novel Cell-Surface Ag Expressed on Rat Osteoclasts Regulating the Function of the Calcitonin Receptor., J Immunology, 153, 11, 5265-5273, 153 (11): 5265-5273, 1995.01.
95. H MAEDA, T KUKITA, A AKAMINE, A KUKITA, T IIJIMA, LOCALIZATION OF OSTEOPONTIN IN RESORPTION LACUNAE FORMED BY OSTEOCLAST-LIKE CELLS - A STUDY BY A NOVEL MONOCLONAL-ANTIBODY WHICH RECOGNIZES RAT OSTEOPONTIN, HISTOCHEMISTRY, 102, 4, 247-254, 102 (4): 247-254, 1994.10, The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface..