Haruka Sato, Tomokazu Hasegawa, Cha-Gyun Jing, Kun Zou, (名) Sato and Makoto Michikawa : Porphyromonas gigivalis Lipopolysaccharide suppres apolipoprotein E secretion from immortalized periodontal ligament fibroblasts derived from human deciduous teeth, 日本歯科保存学雑誌, 69, 2, 135-143, 2026.
(Keyword)
歯根膜線維芽細胞 / アポリポタンパク質E / リポ多糖
3.
Yoshihito Yamakawa, Kimiko Ueda Yamaguchi, Asuna Sugimoto, Yuki Akazawa, Tomokazu Hasegawa, Yumiko Nakashima, Rika Kurogoushi, Manami Tanaka, Nauval Muhammad Dhiaulfikri Hadiana, Nodoka Ato, Akihito Yamamoto, Tomonori Iwasaki and Tsutomu Iwamoto : Ultracentrifuged serum fractions reveal promotion of dental epithelial proliferation by lactoferrin, Journal of Oral Biosciences, 68, 1, 100728, 2026.
(Summary)
Serum is an essential supplement of basal cell culture media, providing growth factors, adhesion molecules, hormones, lipids, and minerals that support cell survival and function. This study was conducted to investigate the effects of ultracentrifugation-separated fractions of fetal bovine serum (FBS) on the proliferation and differentiation of dental epithelial cells with the aim of identifying the key serum-derived factors that regulate cellular activities. FBS was fractionated by ultracentrifugation at 100,000×g for 18 h at 4 °C into three layers, designated as the FBS-1st (top), -2nd (middle), and 3rd (bottom) fractions. Each fraction was added at 10 % (v/v) to the culture medium of rat dental epithelial HAT-7 cells. Cell proliferation was assessed by cell counting and 5-bromo-2'-deoxyuridine incorporation, and differentiation was evaluated by ameloblastin immunostaining. The involvement of lactoferrin and its receptors was examined using recombinant protein stimulation, reverse transcription polymerase chain reaction, small interfering RNA knockdown, and western blotting. FBS-3rd significantly promoted cell proliferation, whereas FBS-1st and -2nd inhibited it. Ameloblastin staining revealed no significant differences between FBS-1st and the control; however, it was suppressed in the FBS-2nd and -3rd. Proteomic analysis identified lactoferrin as the most abundant protein in FBS-3rd, and exogenous lactoferrin enhanced HAT-7 cell proliferation. The lactoferrin receptor, low-density lipoprotein receptor-related protein 1 (Lrp1), was expressed in HAT-7 cells, and Lrp1 knockdown stopped lactoferrin-induced proliferation. Lactoferrin induced phosphorylation of extracellular signal-regulated kinase 1/2, but not Akt serine/threonine kinase, and mitogen-activated protein kinase kinase inhibition with U0126 suppressed the proliferative effect of lactoferrin. Lactoferrin, which was enriched in the FBS-3rd fraction, may contribute to the proliferation of dental epithelial cells via the Lrp1-ERK signaling pathway.
Kimiko Ueda Yamaguchi, Yuki Akazawa, Takamasa Kitamura, Tomokazu Hasegawa, Yoshihito Yamakawa, 鈴木 結加里, 秋田 理沙, Kei Maeo, 幸平 若菜, Hiroshi Nakagawa and Tomonori Iwasaki : Survey of New Patients with Dysphagia at the Pediatric Dentistry Department of Tokushima University Hospital between 2015 and 2022, The Japanese Journal of Pediatric Dentistry, 62, 2, 39-43, 2024.
(Summary)
<p>The Pediatric Feeding and Swallowing Outpatient Clinic provides support for children with delays or abnormalities in feeding and swallowing during their growth period. In this study, 45 children (28 boys and 17 girls) who attended our pediatric dental clinic in the last 7 years and 4 months (from 1 April 2015 to 31 July 2022) were examined at their first visit and the results were tabulated.</p><p>The mean age at first visit was 3 years and 8 months, ranging from 6 months to 11 years and 5 months. Children aged 1 to 3 years accounted for 67% of the total. Twenty-two children were full-term births and 16 were preterm births (including 6 very preterm and 7 extremely preterm). At birth, 25 weighed more than 2,500 g and 19 weighed less than 2,500 g (including 4 less than 1,500 g and 8 less than 1,000 g). Motor development was delayed in 14 children with gross motor development. The most common condition was Down syndrome (11 patients), followed by autism spectrum disorder (7 patients). Fifteen patients were referred from the pediatric department of this hospital. The chief complaints were lack of mastication (14 patients), difficulty in swallowing (8 patients) and difficulty in eating solid food (7 patients). The most common method of feeding was oral intake only (27 patients), followed by oral intake and milk feeding (10 patients), and tube feeding was required in 5 patients. In the future, it is important to continue to provide support for children with feeding and swallowing problems in collaboration with medical, dental and community services, while conveying the importance of attending a medical facility that can provide feeding and swallowing support from an early age.</p>
Anrizandy Narwidina, Aya Miyazaki, Kokoro Iwata, Rika Kurogoushi, Asuna Sugimoto, Yasusei Kudo, Keita Kawarabayashi, Yoshihito Yamakawa, Yuki Akazawa, Takamasa Kitamura, Hiroshi Nakagawa, Kimiko Ueda Yamaguchi, Tomokazu Hasegawa, Keigo Yoshizaki, Satoshi Fukumoto, Akihito Yamamoto, Naozumi Ishimaru, Tomonori Iwasaki and Tsutomu Iwamoto : Iroquois homeobox 3 regulates odontoblast proliferation and differentiation mediated by Wnt5a expression., Biochemical and Biophysical Research Communications, 650, 47-54, 2023.
(Summary)
Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.
Takeyoshi Asakawa, Atsushi Yamada, Masumi Kugino, Tomokazu Hasegawa, Kentaro Yoshimura, Kiyohito Sasa, Mitsuhiro Kinoshita, Masakazu Nitta, Karin Nagata, Tomomi Sugiyama, Ryutaro Kamijo and Takahiro Funatsu : Establishment of Downs syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT, Human Cell, 35, 1, 379-383, 2022.
(Summary)
Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.
Rika Kurogoushi, Tomokazu Hasegawa, Yuki Akazawa, Kokoro Iwata, Asuna Sugimoto, Kimiko Ueda Yamaguchi, Aya Miyazaki, Anrizandy Narwidina, Keita Kawarabayashi, Takamasa Kitamura, Hiroshi Nakagawa, Tomonori Iwasaki and Tsutomu Iwamoto : Fibroblast growth factor 2 suppresses the expression of C-C motif chemokine 11 through the c-Jun N-terminal kinase pathway in human dental pulp-derived mesenchymal stem cells, Experimental and Therapeutic Medicine, 22, 6, 1356, 2021.
(Summary)
The regulation of the mesenchymal stem cell (MSC) programming mechanism promises great success in regenerative medicine. Tissue regeneration has been associated not only with the differentiation of MSCs, but also with the microenvironment of the stem cell niche that involves various cytokines and immune cells in the tissue regeneration site. In the present study, fibroblast growth factor 2 (FGF2), the principal growth factor for tooth development, dental pulp homeostasis and dentin repair, was reported to affect the expression of cytokines in human dental pulp-derived MSCs. FGF2 significantly inhibited the expression of chemokine C-C motif ligand 11 (CCL11) in a time- and dose-dependent manner in the SDP11 human dental pulp-derived MSC line. This inhibition was diminished following treatment with the AZD4547 FGF receptor (FGFR) inhibitor, indicating that FGF2 negatively regulated the expression of CCL11 in SDP11 cells. Furthermore, FGF2 activated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNK) in SDP11 cells. The mechanism of the FGFR-downstream signaling pathway was then studied using the SB203580, U0126 and SP600125 inhibitors for p38 MAPK, ERK1/2, and JNK, respectively. Interestingly, only treatment with SP600125 blocked the FGF2-mediated suppression of CCL11. The present results suggested that FGF2 regulated the expression of cytokines and suppressed the expression of CCL11 via the JNK signaling pathway in human dental pulp-derived MSCs. The present findings could provide important insights into the association of FGF2 and CCL11 in dental tissue regeneration therapy.
岩田 こころ, Rika Kurogoushi, Asuna Sugimoto, 藤島 史帆, Yuki Akazawa, Keita Kawarabayashi, Aya Miyazaki, Takamasa Kitamura, Rie Niji, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa, Tomokazu Hasegawa and Tsutomu Iwamoto : Survey of the Actual State of New Patients at the Pediatric Dentistry Department of Tokushima University Hospital between 1989 and 2018, The Japanese Journal of Pediatric Dentistry, 59, 1, 8-13, 2021.
(Summary)
<p>The Heisei Era ended at the end of April 2019 and the Reiwa Era began.</p><p>For about 30 years in the Heisei era after the Showa era, the environment surrounding children changed rapidly, including the economic downturn, the acceleration of late marriage and declining birthrate, and progress in information and communication technology. As a result, individual households have diversified and a large polarization has become apparent. As pediatric dentists, we need to deal with and support children with all kinds of problems. Therefore, to clarify the transition of the first-visit patients in our clinic, we categorized the 30 years from 1989 to 2018 by five-year groups: early (1989-1993), middle (1997-2001) and late (2013-2018) stages, with a total of 5,892 patients, and then investigated and analyzed their properties. As a result, the number of new patients decreased from 2,477 in the early stage to 1,301 in the late stage. The male-female ratio was approximately 1:1 in each period. As for age, school age was the highest at about 40%, and there was no significant change in the age composition in each period. The most common chief complaint during each period was tooth decay, but it decreased from 44% in the early stage to 27% in the late stage. The proportion of children who brought a letter of referral increased from 23% in the early stage to 83% in the late stage, and the proportion of children with disabilities and illness increased from 13.8% in the early stage to 33% in the late stage. Compared to the early days, the chief complaints have diversified, and it has become clear that the functional differentiation of general dental clinics and university hospitals is progressing. Furthermore, it was suggested that the importance of medical and dental cooperation will increase in the future.</p>
(Keyword)
実態調査 / 初診患者 / 平成 / 小児歯科 / Survey of actual state / New patients / Heisei / Pediatric dentistry
Aya Miyazaki, Asuna Sugimoto, Keigo Yoshizaki, Keita Kawarabayashi, Kokoro Iwata, Rika Kurogohshi, Takamasa Kitamura, Kunihiro Otsuka, Tomokazu Hasegawa, Yuki Akazawa, Satoshi Fukumoto, Naozumi Ishimaru and Tsutomu Iwamoto : Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1, Scientific Reports, 9, 1, 14762, 2019.
(Summary)
Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.
Kimiko Ueda Yamaguchi, Yuki Akazawa, Kawarabayashi Keita, Asuna Sugimoto, Hiroshi Nakagawa, miyazaki aya, Weih Falk, Kurogoushi Rika, Iwata Kokoro, Takamasa Kitamura, Yamada Aya, Tomokazu Hasegawa, Fukumoto Satoshi and Tsutomu Iwamoto : Combination of ions promotes cell migration via extracellular signalregulated kinase 1/2 signaling pathway in human gingival fibroblasts., Molecular Medicine Reports, 19, 6, 5039-5045, 2019.
(Summary)
Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre-reacted glass-ionomer (S-PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S-PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S-PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF-1 was treated with various dilutions of an eluent solution of S-PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF-1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S-PRG solution and revealed that it activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S-PRG fillers can stimulate HGF-1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing.
Kifune Takashi, Ito Hisanori, Ishiyama Misa, Iwasa Satoko, Takei Hiroki, Tomokazu Hasegawa, Asano Masatake and Tetsuo Shirakawa : Hypoxia-induced up-regulation of angiogenic factors in immortalized human periodontal ligament fibroblasts, Journal of Oral Science, 60, 4, 519-525, 2018.
(Summary)
Hypoxia induces complex cellular responses that are mediated by a key transcription factor, hypoxia-inducible factor-1 (HIF-1). HIF-1 promotes production of cytokines and angiogenic factors and contributes to recovery of injured tissues. In the present study, expressions of angiogenin (ANG) and vascular endothelial growth factor (VEGF), which are potent angiogenic factors in mammalian tissues, were examined in immortalized fibroblasts exposed to hypoxia. After 24 h of exposure to hypoxia, ANG and VEGF mRNAs expressions were significantly elevated in periodontal ligament (PDL) fibroblasts but not in embryonic fibroblasts. Hypoxia also increased productions of ANG and VEGF proteins in PDL fibroblasts. HIF-1α mRNA expression was not affected by hypoxia in either fibroblast, although HIF-1α protein expression was enhanced after exposure to hypoxia. Treatment of PDL fibroblasts with dimethyloxaloylglycine, a prolyl hydroxylase inhibitor that stabilizes the HIF-1α protein, significantly increased expressions of ANG and VEGF mRNAs under normoxia. This suggests that stabilization of HIF-1α is crucial for upregulation of ANG and VEGF in PDL fibroblasts. These results indicate that, under hypoxic conditions, HIF-1α upregulates synthesis of ANG and VEGF in PDL fibroblasts and promotes angiogenesis.
Ito Hisanori, Kifune Takashi, Ishiyama Misa, Iwasa Satoko, Takei Hiroki, Tomokazu Hasegawa, Asano Masatake and Shirakawa Tetsuo : Effect of hypoxia on the expression of CCAAT/enhancer-binding protein β and receptor activator of NF-κB ligand in periodontal ligament cells, Journal of Oral Science, 60, 4, 544-551, 2018.
(Summary)
Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-β (C/EBPβ) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPβ and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1β, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPβ and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPβ and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPβ gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPβ may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.
Asuna Sugimoto, Aya Miyazaki, Keita Kawarabayashi, Masayuki Shono, Yuki Akazawa, Tomokazu Hasegawa, Kimiko Ueda Yamaguchi, Takamasa Kitamura, Yoshizaki keigo, Fukumoto Satoshi and Tsutomu Iwamoto : Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells, Scientific Reports, 7, 1, 17696, 2017.
(Summary)
The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.
Yoshitaka Yoshimura, Takashi Kikuiri, Tomokazu Hasegawa, Mino Matsuno, Hajime Minamikawa, Yoshiaki Deyama and Kuniaki Suzuki : How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro, Molecular Medicine Reports, 16, 1, 429-434, 2017.
(Summary)
Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24-well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume-dependent manner. A co-culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP-positive multinuclear cells (≥8 nuclei) was increased, in a media volume-dependent manner. There were also no differences in receptor activator of nuclear factor-κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume-dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.
Nan Ma, Di Yang, Hirohiko Okamura, Jumpei Teramachi, Tomokazu Hasegawa, Lihong Qiu and Tatsuji Haneji : Involvement of interleukin23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis, Molecular Medicine Reports, 15, 2, 559-566, 2017.
(Summary)
Periapical lesions are characterized by the destruction of periapical bone, and occur as a result of local inflammatory responses to root canal infection by microorganisms including Porphyromonas endodontalis (P. endodontalis). P. endodontalis and its primary virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical lesions and alveolar bone loss. Interleukin-23 (IL-23) is critical in the initiation and progression of periodontal disease via effects on peripheral bone metabolism. The present study investigated the expression of IL-23 in tissue where a periapical lesion was present, and the effect of P. endodontalis LPS on the expression of IL-23 in periodontal ligament (PDL) cells. Reverse transcription- quantitative polymerase chain reaction and immunohistochemistry revealed increased levels of IL-23 expression in tissue with periapical lesions compared with healthy PDL tissue. Treatment with P. endodontalis LPS increased the expression of IL-23 in the SH-9 human PDL cell line. BAY11-7082, a nuclear factor κB inhibitor, suppressed P. endodontalis LPS-induced IL-23 expression in SH-9 cells. Treatment of RAW264.7 cells with conditioned medium from P. endodontalis LPS-treated SH-9 cells promoted osteoclastogenesis. By contrast, RAW264.7 cells treated with conditioned medium from IL-23-knockdown SH-9 cells underwent reduced levels of osteoclastogenesis. The results of the present study indicated that the expression of IL-23 in PDL cells induced by P. endodontalis LPS treatment may be involved in the progression of periapical lesions via stimulation of the osteoclastogenesis process.
Masaaki Ikeda, Yoshitaka Yoshimura, Takashi Kikuiri, Mino Matsuo, Tomokazu Hasegawa, Kumu Fukushima, Takako Hayakawa, Hajime Minamikawa, Kuniaki Suzuki and Junichiro Iida : Release from optimal compressive force suppresses osteoclast differentiation, Molecular Medicine Reports, 14, 5, 4699-4705, 2016.
(Summary)
Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription-quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast-specific genes, TRAP, RANK, matrix metalloproteinase-9, cathepsin-K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell-specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC-STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.
Takako Hayakawa, Yoshitaka Yoshimura, Takashi Kikuiri, Mino Matsuno, Tomokazu Hasegawa, Kumu Fukushima, Kenjiro Shibata, Yoshiaki Deyama, Kuniaki Suzuki and Junichiro Iida : Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells, Molecular Medicine Reports, 12, 4, 5879-5885, 2015.
(Summary)
Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast-associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.
Yuki Akazawa, Tomokazu Hasegawa, Yoshitaka Yoshimura, Naoyuki Chosa, Takeyoshi Asakawa, Kimiko Ueda Yamaguchi, Asuna Sugimoto, Takamasa Kitamura, Hiroshi Nakagawa, Akira Ishisaki and Tsutomu Iwamoto : Recruitment of mesenchymal stem cells by stromal cell-derived factor 1 in pulp cells from deciduous teeth, International Journal of Molecular Medicine, 36, 442-448, 2015.
(Summary)
Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1 (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.
Tomokazu Hasegawa, Yuki Akazawa, Takamasa Kitamura, Asuna Sugimoto, Kimiko Ueda Yamaguchi and Tsutomu Iwamoto : Dental findings and management in a child with hypomelanosis of Ito, Pediatric Dental Journal, 24, 3, 173-177, 2014.
Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy.
(Keyword)
ADP-ribosyl Cyclase / Antigens, CD / Cell Differentiation / Cell Movement / Down-Regulation / Extracellular Signal-Regulated MAP Kinases / Focal Adhesion Protein-Tyrosine Kinases / GPI-Linked Proteins / Gene Expression Regulation, Developmental / Humans / JNK Mitogen-Activated Protein Kinases / Mesenchymal Stromal Cells / Nerve Tissue Proteins / Osteogenesis / Phosphatidylinositol 3-Kinases / Protein Binding / Protein Biosynthesis / Signal Transduction
Tomokazu Hasegawa, Yuki Akazawa, Hirokazu Nagai, Takamasa Kitamura, Naozumi Ishimaru, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa, Yukiko Kori, Aimi Yamamoto and Tsutomu Iwamoto : Capillary Hemangioma on Palate of Infant:Case Report, The Japanese Journal of Pediatric Dentistry, 52, 3, 448-453, 2014.
(Summary)
A hemangioma frequently occurs on the tongue, lip, buccal mucosa, and gingiva in the oral region,though it relatively rarely seen on the palate. We report a case of capillary hemangioma found on the palate of a 2-year-10-month-old boy. He was referred to our department with the chief complaint of a tumor on the palate and a hemangioma was noted on the palatal side of the upper deciduous incisors.We performed surgical resection and the mass was extirpated. The postoperative course was uneventful. Although there was no evidence of recurrence at 6 months after surgery, careful follow-up examinations will be necessary until the upper permanent incisors erupt.
Jun Yokota, Jun Yokota, Shunsuke Sawada, Naoto Okubo, Noriko Takahashi, Tomokazu Hasegawa, Hisatomo Kondo and Akira Ishisaki : PDGF-induced PI3K-mediated signal enhances TGF--induced osteogenic differentiation of human mesenchymal stem cells in the TGF--activated MEK-dependent manner, International Journal of Molecular Medicine, 33, 3, 534-542, 2013.
(Summary)
Transforming growth factor-β (TGF-β) is a critical regulator of osteogenic differentiation and the platelet-derived growth factor (PDGF) is a chemoattractant or mitogen of osteogenic mesenchymal cells. However, the combined effects of these regulators on the osteogenic differentiation of mesenchymal cells remains unknown. In this study, we investigated the effects of TGF-β and/or PDGF on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The TGF-β-induced osteogenic differentiation of UE7T-13 cells, a bone marrow-derived hMSC line, was markedly enhanced by PDGF, although PDGF alone did not induce differentiation. TGF-β induced extracellular signal-regulated kinase (ERK) phosphorylation and PDGF induced Akt phosphorylation. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, suppressed the osteogenic differentiation induced by TGF-β alone. Moreover, U0126 completely suppressed the osteogenic differentiation synergistically induced by TGF-β and PDGF, whereas the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002, only partially suppressed this effect. These results suggest that the enhancement of TGF-β-induced osteogenic differentiation by PDGF-induced PI3K/Akt-mediated signaling depends on TGF-β-induced MEK activity. Thus, PDGF positively modulates the TGF-β-induced osteogenic differentiation of hMSCs through synergistic crosstalk between MEK- and PI3K/Akt-mediated signaling.
Yuki Akazawa, Takamasa Kitamura, Yuri Fujihara, Yoshitaka Yoshimura, Masato Mitome and Tomokazu Hasegawa : Forced mastication increases survival of adult neural stem cells in the hippocampal dentate gyrus., International Journal of Molecular Medicine, 31, 2, 307-314, 2012.
(Summary)
In this study, we examined the effect of forced mastication on neurogenesis in the hippocampal dentate gyrus (DG) of adult mice. Six-week-old mice were subjected to either a hard or normal diet for 13 weeks. They received a daily injection of bromodeoxyuridine (BrdU) for 12 consecutive days beginning at 14 weeks of age. The number of BrdU-positive cells in the DG was counted 1 day after and 5 weeks after the final BrdU injection. The number of BrdU-positive cells 1 day after injection did not differ between the 2 diet groups. However, the number of BrdU-positive cells in the group fed the hard diet was significantly increased 5 weeks after BrdU injection compared to the group fed the normal diet. The results of the Morris water maze test showed that mice fed a hard diet required significantly less time to reach the platform than the control mice when tested at 10 days. Moreover, mice in the group fed the hard diet spent significantly more time in the former platform area than the group fed the normal diet, indicating that hard diet feeding improved spatial memory compared to normal diet feeding. Real-time PCR analysis showed that the expression of glutamate receptor 1 mRNA was significantly increased in the group fed the hard diet compared with the group fed the normal diet. These results suggest that mastication increases the survival of adult neural stem cells in the hippocampal DG.
Mariko Yoshida, Naoto Okubo, Naoyuki Chosa, Tomokazu Hasegawa, Miho Ibi, Masaharu Kamo, Seiko Kyakumoto and Akira Ishisaki : TGF-β-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals., International Journal of Biological Sciences, 8, 7, 1062-1074, 2012.
(Summary)
The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.
Tomokazu Hasegawa, Naoyuki Chosa, Takeyoshi Asakawa, Yoshitaka Yoshimura, Yuri Fujihara, Takamasa Kitamura, Mitsuro Tanaka, Akira Ishisaki and Masato Mitome : Differential effects of TGF-β1 and FGF-2 on SDF-1α expression in human periodontal ligament cells derived from deciduous teeth in vitro., International Journal of Molecular Medicine, 30, 1, 35-40, 2012.
(Summary)
Stromal cell-derived factor (SDF)-1α has been reported to play a crucial role in stem cell homing and recruitment to injured sites. However, no information is available about its role in periodontal tissues. The aim of this in vitro study was to investigate the effects of basic fibroblast growth factor (FGF-2) and transforming growth factor (TGF)-β1 on SDF-1α expression in immortalized periodontal ligament (PDL) cells derived from deciduous teeth (SH9 cells). Real-time PCR and western blot analyses showed that SDF-1α mRNA expression in SH9 cells was markedly inhibited by FGF-2 treatment for 48 h. SU5402, which directly interacts with the catalytic domain of the FGF receptor 1 (FGFR1) and suppresses its phosphorylation, inhibited the FGF-2-related decrease in SDF-1α expression. These results suggest that FGF-2 signaling via the FGFR1 pathway inhibits SDF-1α expression. Conversely, SDF-1α expression in SH9 cells was increased by TGF-β1 treatment for 12 h. Western blot analysis showed that this treatment induced Smad2/3 phosphorylation. A time-course experiment showed that SDF-1α expression levels reached a maximum 12 h after the TGF-β1 treatment and returned to basal levels by 48 h. Real-time PCR analysis showed that Smad7 mRNA expression peaked by 6 h after TGF-β1 treatment. Since Smad7 siRNA downregulated Smad7 expression by approximately 2.5-fold compared with the negative control siRNA, the induction of SDF-1α expression was prolonged. Furthermore, treatment of SH9 cells with TGF-β1 for 12 h induced transwell migration of UE7T-13 cells, which are mesenchymal stem cells derived from human bone marrow. Therefore, SDF-1α may play an important role in stem and progenitor cell recruitment and homing to injured sites in the periodontal ligament, and regulation of SDF-1α expression may be a useful tool in cell-based therapy for periodontal tissue regeneration.
(Keyword)
Bone Marrow Cells / Cell Line / Cell Movement / Chemokine CXCL12 / Fibroblast Growth Factor 2 / Humans / Mesenchymal Stromal Cells / Periodontal Ligament / Phosphorylation / Pyrroles / RNA Interference / RNA, Messenger / RNA, Small Interfering / Receptors, Fibroblast Growth Factor / Smad2 Protein / Smad3 Protein / Smad7 Protein / Tooth, Deciduous / Transforming Growth Factor beta1
Keigo Abe, Yoshitaka Yoshimura, Yoshiaki Deyama, Tkashi Kikuiri, Tomokazu Hasegawa, Kanchu Tei, Hisashi shinoda, Kuniaki Suzuki and Yoshimasa Kitagawa : Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells., International Journal of Molecular Medicine, 29, 6, 1007-1015, 2012.
(Summary)
Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.
Yuri Fujihara, 赤澤 友基, Takamasa Kitamura, Tomokazu Hasegawa and Masato Mitome : Many Congenital Missing Teeth in a Case of Bloch-Sulzberger Syndrome, Journal of the Japanese Society for Disability and Oral Health, 33, 1, 74-79, 2012.
Takeyoshi Asakawa, Naoyuki Chosa, Yoshitaka Yoshimura, Asami Asakawa, Mitsuro Tanaka, Akira Ishisaki, Masato Mitome and Tomokazu Hasegawa : Fibroblast growth factor 2 inhibits the expression of stromal cell-derived factor 1 in periodontal ligament cells derived from human permanent teeth in vitro., International Journal of Molecular Medicine, 29, 4, 569-573, 2012.
(Summary)
Although cells derived from periodontal ligament (PDL) tissue are reported to have stem cell-like activity and are speculated to play a crucial role for tissue healing and regeneration after injury or orthodontic treatment, mechanisms regulating their recruitment and activation remain unknown. Recently, stromal cell-derived factor 1α (SDF-1α) has been reported to be important for stem cell homing and recruitment to injured sites. The aim of this study was to evaluate whether fibroblast growth factor 2 (FGF-2) affects the expression of SDF-1α in PDL cells derived from human permanent teeth in vitro. Using real-time PCR, the expression of SDF-1α mRNA in PDL cells was inhibited by treatment with 10 ng/ml FGF-2. When PDL cells were treated with SU5402 (an inhibitor of FGF receptor 1) in combination with FGF-2, the FGF-2-reduced expression of SDF-1α was inhibited. In the presence of the JNK inhibitor SP600125, SDF-1α mRNA in PDL cells was not suppressed by the FGF-2 treatment. Western blot analysis also showed that SDF-1α production was suppressed by treatment with FGF-2, but it recovered with treatment by FGF-2 + SU5402. These findings suggest that SDF-1α from PDL cells plays an important role in the regeneration and homeostasis of periodontal tissues via the recruitment of stem cells.
Mayumi Nomura, Yoshitaka Yoshimura, Takashi Kikuiri, Tomokazu Hasegawa, Yumi Taniguchi, Yoshiaki Deyama, Ken-ichi Koshiro, Hidehiko Sano, Kuniaki Suzuki and Nobuo Inoue : Platinum nanoparticles suppress osteoclastogenesis through scavenging of ROS productions in RAW264.7 cells., Journal of Pharmacological Sciences, 117, 4, 243-252, 2011.
(Summary)
Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOX-family oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.
Takashi Kikuiri, Yoshitaka Yoshimura, Futoshi Tabata, Tomokazu Hasegawa, Jun Nishihira and Tetsuo Shirakawa : Stage-dependent suppression of the formation of dentin-resorbing multinuclear cells with migration inhibitory factor in vitro., Experimental and Therapeutic Medicine, 3, 1, 37-43, 2011.
(Summary)
The macrophage migration inhibitory factor (MIF) is a crucial mediator of immune responses and is known to play a pivotal role in cell proliferation and differentiation. In this study, we assessed whether MIF exerts regulatory effects on osteoclast formation in bone marrow cells and, if so, by what mechanism. Bone marrow cells were either co-cultured with MC3T3-E1 cells or cultured with macrophage-colony stimulating factor (M-CSF) and the soluble form of the receptor activator of the nuclear factor-B ligand (RANKL). Under the influence of MIF, the formation of osteoclastic multinuclear cells was examined. The number of multinuclear TRAP-positive cells formed in the co-culture was significantly reduced when MIF (0.1 g/ml) was exogenously applied during the third and fourth days of the 6-day cultivation period. MIF affected neither the number of mononuclear TRAP-positive cells induced with M-CSF and RANKL, nor the expression of RANKL and osteoprotegerin in MC3T3-E1 cells. TRAP-positive cells cultured on dentin slices with MIF showed lower dentin-resorbing activity than those cultured without MIF. These results suggest that MIF has no regulatory roles in the differentiation of bone marrow cells to mononuclear TRAP-positive cells, but has inhibitory effects on the formation of mature osteoclasts by preventing cell fusion, which may eventually interfere with the osteoclast-mediated dentin resorption.
Noriko Takahashi, Naoyuki Chosa, Tomokazu Hasegawa, Soko Nishihira, Naoto Okubo, Mamoru Takahashi, Yoshiki Sugiyama, Mitsuro Tanaka and Akira Ishisaki : Dental pulp cells derived from permanent teeth express higher levels of R-cadherin than do deciduous teeth: implications of the correlation between R-cadherin expression and restriction of multipotency in mesenchymal stem cells., Archives of Oral Biology, 57, 1, 44-51, 2011.
(Summary)
The aim of this study was to characterize the expression status of cadherins in dental pulp-derived mesenchymal progenitor/stem cells from deciduous and permanent teeth, and to determine how cadherins affect the multipotency of the progenitor/stem cells. We evaluated and compared the expression status of cadherins in dental pulp-derived cells from deciduous teeth and in cells from permanent teeth by using an array of primers for amplification of RNA encoding human cell adhesion molecules and a real time PCR system. In order to elucidate how cadherins (which are differentially expressed in deciduous and permanent teeth) affect the multipotency of the dental pulp-derived progenitor/stem cells, the ability of the dental pulp cells to differentiate into adipocytes and osteoblasts was evaluated. R-cadherin was found to be vigorously expressed in the dental pulp cells derived from permanent teeth but not in the dental pulp cells derived from deciduous teeth. N-cadherin was found to be expressed essentially equally in both types of cells. The ability of the dental pulp cells of deciduous teeth to differentiate into adipocytes and osteoblasts was found to be much higher than that of cells obtained from permanent teeth. R-cadherin may be a key molecule for providing control over the multipotency of the dental pulp-derived mesenchymal stem cells.
Kenjiro Shibata, Yoshitaka Yoshimura, Takashi Kikuiri, Tomokazu Hasegawa, Yumi Taniguchi, Yoshiaki Deyama, Kuniaki Suzuki and Junichiro Iida : Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells., International Journal of Molecular Medicine, 28, 1, 73-79, 2011.
(Summary)
The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.
Tomokazu Hasegawa, Naoyuki Chosa, Takeyoshi Asakawa, Yoshitaka Yoshimura, Akira Ishisaki and Mitsuro Tanaka : Eatablishment of periodontal ligament cell line derived from human deciduous teeth, Interface oral health science 2011, 114-116, 2012.
Review, Commentary:
1.
Kimiko Ueda Yamaguchi, Keita Kawarabayashi, Aya Miyazaki, Rika Kurogohshi, Kokoro Iwata, Asuna Sugimoto, Yuki Akazawa, Tomokazu Hasegawa and Tsutomu Iwamoto : Prevention of Sports-related Dental Injuries in Children, Journal of Oral Health and Biosciences, 31, 1, 68-72, Jul. 2018.
(Summary)
A sports-related dental injury is defined as injuries to the oral and maxillofacial regions associated with sports activities, and involves tooth fracture and luxation, facial bone and temporomandibular fractures, and soft tissue injury. Participants in sports activities are always at risk for traumatic injury, with the oral and maxillofacial region often affected. Dental injuries also have a high rate of occurrence among sports-related injuries received during school physical education classes and club activities. Unfortunately, nearly all such dental injuries are irreversible, and the loss of teeth or their supporting tissues has a significant impact on the quality of life of affected individuals. Thus, for prevention of sports-related dental injuries, it is important for dental professionals to disseminate correct knowledge regarding oral health, as well as provide information to reduce and treat risk factors such as dental caries, periodontal disease, and occlusal problems. In particular, use of mouthguard is one of the most effective ways to prevent sports-related dental injuries that occur in sports and physical activity participants. Recently, along with increased health consciousness, the number of individuals who participate in sports and fitness activities is also increasing. Outside of the bounds of conventional dental clinical treatment, dentists are encouraged to actively be involved in local and regional organizations related to sports, recreation, and physical activity opportunities, in order to contribute to promotion of safety and health, including injury prevention. In this review, we discuss various findings to prevention of sports-related dental injuries in children.
Tsutomu Iwamoto, Asuna Sugimoto, Takamasa Kitamura, Yuki Akazawa and Tomokazu Hasegawa : The role of extracellular ATP-mediated purinergic signaling in bone, cartilage, and tooth tissue, Journal of Oral Biosciences, 56, 4, 131-135, Jul. 2014.
Yamakawa Yoshihito, Yuki Akazawa, Tomokazu Hasegawa, Takamasa Kitamura, Kei Maeo, Kimiko Ueda Yamaguchi, Fumiya Kano, Takaaki Tsunematsu, Hiroshi Nakagawa, Tomonori Iwasaki and Tsutomu Iwamoto : A case of narrowing of dental pulp cavity on deciduous teeth 10-year-old boy long-term treated by steroid, International association of pediatric dentistry 2025, 22-25, 2025.
2.
Yamakawa Yoshihito, Kimiko Ueda Yamaguchi, Akazawa Yuki, Tomokazu Hasegawa, Nakashima Yumiko, Iwata Kokoro, Asuna Sugimoto, Tomonori Iwasaki and Tsutomu Iwamoto : Analysis of serum components to find and identify factor that promotes cell differentiation in. dental epithelial cells, pediatric dentistry association of Aisa, Oct. 2024.
3.
Takeyoshi Asakawa, Yoichi Miyamoto, Kentaro Yoshimura, Kiyoto Sasa, Yoko Manome, Ryutaro Kamijo, Tomokazu Hasegawa, Naoyuki Chosa, Akira Ishizaki, Masumi Ookawa, Tomomi Sugiyama, Masayasu Shige and Yukie Shimada : Establish of expression on differentiation periodontal ligament cells derived from human teeth-Cooparative analysis that SDF-1 regulation of expression on periodontal ligament cells derived from Down syndrome teeth, 116, 2017.
4.
Kimiko Ueda Yamaguchi, Asuna Sugimoto, Yuki Akazawa, Tomokazu Hasegawa and Tsutomu Iwamoto : Multi-ion solution promotes cell migration through ERK signaling pathway and CXCR4 expression in human gingival of fibroblasts, 10th Biennial Conferrence of the Pediatric Dentistry Association of Asia, 54, 2, 346, Tokyo, May 2016.
5.
Takeyoshi Asakawa, Yoichi Miyamoto, Kentaro Yoshimura, Sasa Kiyoto, Tomokazu Hasegawa, Naoyuki Chosa, Akira Ishizaki, Miki Kadena, Yoko Manome, Miku Kuritani, Ryutaro Kamijo and Takahiro Funatsu : Establishing and SDF-1 regulation of expression on periodontal ligament cells derived from human teeth, The Japanese Journal of Pediatric Dentistry, 53, 2, 354, May 2016.
(Keyword)
SDF-1 / periodontal ligament / human / permanent teeth
6.
Takeyoshi Asakawa, Naoyuki Chosa, Tomokazu Hasegawa, Asami Asakawa, Akira Ishisaki and Mitsuro Tanaka : Regulation of SDF-1 expression in periodontal ligament cells derived from human permanent teeth., Interface oral health science 2011, 107-109, 2012.
Yuki Akazawa, Tomokazu Hasegawa, Yoshihito Yamakawa, Kimiko Ueda and Tomonori Iwasaki : 圧力下における乳歯歯髄細胞のSDF-1発現解析, The Japanese Journal of Pediatric Dentistry, 64, 342, May 2026.
(Keyword)
ヒト乳歯歯髄細胞 / SDF-1 / 圧迫力
2.
Yuki Akazawa, Tomokazu Hasegawa, YAMAKAWA Yoshihito, Kimiko Ueda Yamaguchi and Tomonori Iwasaki : 圧力下における乳歯歯髄細胞のSDF-1発現の解析, 第44回日本小児歯科学会中四国地方会大会, Nov. 2025.
Yuki Akazawa, Kei Maeo, 蓮井 義則, Wakana Kohira, Yoshihito Yamakawa, 鈴木 結加里, 秋田 理沙, Eri Mizumura, Kimiko Ueda Yamaguchi, Takamasa Kitamura, Yumika Ida, Tomokazu Hasegawa and Tomonori Iwasaki : A Case Report: External Resorption of lower lateral incisor, 第62回日本小児歯科学会, May 2024.
6.
Yuki Akazawa, Tomokazu Hasegawa and Tomonori Iwasaki : 多数の永久歯に著しい短根を認めた1例, The Japanese Journal of Pediatric Dentistry, 62, 74, 2024.
7.
Kimiko Ueda Yamaguchi, Yuki Akazawa, Keita Kawarabayashi, Aya Miyazaki, Takamasa Kitamura, Tomokazu Hasegawa, Hiroshi Nakagawa and Tomonori Iwasaki : 当院小児摂食嚥下外来における初診時調査, The Japanese Journal of Pediatric Dentistry, 61, 53-54, Feb. 2023.
8.
Rika Kurogoushi, Tomokazu Hasegawa, Tomonori Iwasaki and Tsutomu Iwamoto : 両側性に生じた下顎第一大臼歯生歯困難の1例, 日本小児歯科学会, 60, 207, May 2022.
9.
Tomonori Iwasaki, Kei Maeo, Wakana Kohira, Takamasa Kitamura, Keita Kawarabayashi, Aya Miyazaki, Yuki Akazawa, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa and Tomokazu Hasegawa : 小児閉塞性睡眠時無呼吸への対応と展望, 徳島県小児保健協会 総会並びに第62回講演会, 3, Sep. 2021.
10.
黒厚子 璃佳, Tomokazu Hasegawa, Yuki Akazawa and Tsutomu Iwamoto : 歯髄細胞におけるFGF2によるJNK経路を介したCCL11の発現抑制, The Japanese Journal of Pediatric Dentistry, 59, 117, Jun. 2021.
11.
Aya Miyazaki, Tomokazu Hasegawa and Tsutomu Iwamoto : 乳歯の早期萌出をみる ADAM17 欠損症の1例, The Japanese Journal of Pediatric Dentistry, 58, 20, Jun. 2020.
Rika Kurogohshi, Tomokazu Hasegawa, Yuki Akazawa, Asuna Sugimoto, Keita Kawarabayashi, Kimiko Ueda Yamaguchi and Tsutomu Iwamoto : 乳歯歯髄細胞におけるExotaxin-1の発現調節機構の解析, The Japanese Journal of Pediatric Dentistry, 56, 2, 215, May 2018.
20.
浅川 剛吉, 大川 真澄, 永田 夏琳, Tomokazu Hasegawa, 宮本 洋一, 吉村 健太, 笹 清人, 馬目 瑤子, 杉山 智美, 上條 竜太郎 and 島田 幸恵 : Down症候群乳歯歯根膜由来細胞のDYRK1A発現解析, The Japanese Journal of Pediatric Dentistry, 56, 2, 211, May 2018.
21.
黒厚子 璃佳, Yuki Akazawa, Tomokazu Hasegawa, Asuna Sugimoto, Kimiko Ueda Yamaguchi and Tsutomu Iwamoto : 乳歯歯髄細胞におけるケモカインCCL11の発現解析, The Japanese Journal of Pediatric Dentistry, 56, 1, 154, Nov. 2017.
22.
宮嵜 彩, Asuna Sugimoto, 井上 秀人, Takamasa Kitamura, Kimiko Ueda Yamaguchi, 河原林 啓太, Yuki Akazawa, Tomokazu Hasegawa and Tsutomu Iwamoto : 当科における10年間の口唇口蓋裂児の実態調査, 第55回日本小児歯科学会大会, 55, 2, 183, May 2017.
23.
林 光一, 村澤 瑛里子, Tomokazu Hasegawa and Tsutomu Iwamoto : ヒト乳歯歯髄細胞によるSDF-1αの発現はTGF-βを介してFGF-2により負に制御されている, Journal of Oral Health and Biosciences, Mar. 2017.
24.
杉本 明日菜, Yuki Akazawa, Tomokazu Hasegawa, 宮嵜 彩, 河原林 啓太 and Tsutomu Iwamoto : PIEZO1は歯髄細胞の分化運命決定に関与し，象牙芽細胞への分化を促進させる, The Japanese Journal of Pediatric Dentistry, 55, 2, 290, 2017.
25.
木舩 崇, 伊藤 寿典, 石山 未紗, 武井 浩樹, Tomokazu Hasegawa and Tetsuo Shirakawa : 低酸素暴露が株化歯根膜細胞の血管新生因子産生に及ぼす影響, The Japanese Journal of Pediatric Dentistry, 55, 2, 287, 2017.
26.
Yuki Akazawa, Tomokazu Hasegawa, 帖佐 直幸, 吉村 善隆, 浅川 剛吉, 杉本 明日菜, 河原林 啓太, 宮嵜 彩, 石崎 明 and Tsutomu Iwamoto : ヒト乳歯歯髄細胞のSDF-1発現調節機構におけるALK5の関与について, The Japanese Journal of Pediatric Dentistry, 55, 2, 291, 2017.
27.
Yuki Akazawa, Kimiko Ueda Yamaguchi, Tomokazu Hasegawa and Tsutomu Iwamoto : 低位下顎左側第二乳臼歯の再萌出を認めた1例, The Japanese Journal of Pediatric Dentistry, 55, 1, 141-142, Nov. 2016.
28.
Rie Niji, Kimiko Ueda Yamaguchi, Yuki Akazawa, Takamasa Kitamura, Tomokazu Hasegawa, 杉本 明日菜 and Tsutomu Iwamoto : 歯の萌出時期に低濃度フッ素化合物を使用した小児の齲蝕罹患状況, The Japanese Journal of Pediatric Dentistry, 54, 1, 144, Oct. 2015.
29.
Kimiko Ueda Yamaguchi, Yuki Akazawa, Takamasa Kitamura, 杉本 明日菜, Tomokazu Hasegawa, Hiroshi Nakagawa and Tsutomu Iwamoto : ヒト歯肉線維芽細胞におけるS-PRGフィラー抽出液による細胞遊走の促進, The Japanese Journal of Pediatric Dentistry, 54, 1, 145, Oct. 2015.
30.
Kimiko Ueda Yamaguchi, 益富 由佳子, Rie Niji, Takamasa Kitamura, Aimi Yamamoto, Yuki Akazawa, 杉本 明日菜, 白石 真紀, Hiroshi Nakagawa, Tomokazu Hasegawa, Yukiko Kori and Tsutomu Iwamoto : Clinical survey of traumatic dental injuries in children at our outpatient clinic, The Japanese Journal of Pediatric Dentistry, 53, 2, 272, May 2015.
31.
浅川 剛吉, 宮本 洋一, 吉村 健太郎, Tomokazu Hasegawa, 帖佐 直幸, 石崎 明, 山下 一恵, 嘉手納 未季, 馬目 瑤子, 栗谷 未来, 上條 竜太郎 and 船津 敬弘 : ヒトDown症候群歯根膜由来細胞におけるSDF-1α発現解析, The Japanese Journal of Pediatric Dentistry, 53, 2, 265, 2015.
32.
Yuki Akazawa, Tomokazu Hasegawa and Tsutomu Iwamoto : 象牙質修復に関わる間葉系幹細胞の乳歯歯髄細胞由来SDF-1αによる制御, The Japanese Journal of Pediatric Dentistry, 53, 2, 221, 2015.
33.
Tomokazu Hasegawa, Yuki Akazawa, 吉村 善隆, 帖佐 直幸, 浅川 剛吉, 杉本 明日菜, Takamasa Kitamura, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa, 白石 真紀, 石崎 明 and Tsutomu Iwamoto : 乳歯歯根膜由来細胞のSDF-1αを介した歯周組織恒常性に関わる細胞間相互作用, The Japanese Journal of Pediatric Dentistry, 53, 2, 222, 2015.
34.
幸江 真杉, Yuki Akazawa, Tomokazu Hasegawa and Tsutomu Iwamoto : 先天性気管狭窄症に対する口腔内衛生管理の1例, The Japanese Journal of Pediatric Dentistry, 53, 1, 168, 2015.
35.
浅川 剛吉, 宮本 洋一, 吉村 健太郎, Tomokazu Hasegawa, 山下 一恵, 嘉手納 美季, 馬目 瑤子, 栗谷 未来, 上条 竜太郎 and 船津 敬弘 : ヒトDown症乳歯歯根膜細胞のSDF-1発現調節の解析, Journal of the Japanese Society for Disability and Oral Health, 35, 3, 278, 2014.
Hiroshi Nakagawa, Aimi Yamamoto, Yuki Akazawa, Takamasa Kitamura, Tomokazu Hasegawa and Tsutomu Iwamoto : p-HPPHを分子標的としたフェニトインによる歯肉増殖症に対する新規治療薬の開発, Journal of the Japanese Society for Disability and Oral Health, 35, 3, 478, 2014.
39.
Takamasa Kitamura, Yukiko Kori, 赤澤 友基, Tomokazu Hasegawa and Tsutomu Iwamoto : 鼻腔底に近接した上顎正中埋伏過剰歯由来の含歯性嚢胞の1例, The Japanese Journal of Pediatric Dentistry, 52, 2, 306, 2014.
40.
赤澤 友基, Tomokazu Hasegawa, 久保-藤原 百合, Takamasa Kitamura, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa and Tsutomu Iwamoto : 当科にて長期的口腔内管理を行った伊藤白斑の1例, The Japanese Journal of Pediatric Dentistry, 52, 2, 354, 2014.
41.
Tomokazu Hasegawa, 赤澤 友基, Takamasa Kitamura, Kimiko Ueda Yamaguchi, Hiroshi Nakagawa and Tsutomu Iwamoto : 幼児期の小児の口蓋に発生した血管腫の1例, The Japanese Journal of Pediatric Dentistry, 52, 2, 355, 2014.
42.
Tomokazu Hasegawa, 赤澤 友基, Hiroshi Nakagawa, 上田 公子 and Tsutomu Iwamoto : 乳歯歯髄細胞による細胞走化性因子の調節機構, The Japanese Journal of Pediatric Dentistry, 52, 1, 125-126, 2014.
Hiroshi Nakagawa, Aimi Yamamoto, Yukiko Kori, Kimiko Ueda Yamaguchi, 赤澤 友基, Takamasa Kitamura, Tomokazu Hasegawa and Tsutomu Iwamoto : The longitudinal changes in the results of psychological tests applied to a patient with dental phobia, Journal of the Japanese Society for Disability and Oral Health, 34, 3, 375, Oct. 2013.
Hiroshi Nakagawa, Aimi Yamamoto, Yukiko Kori, Kimiko Ueda Yamaguchi, Yuki Akazawa, Takamasa Kitamura, Tomokazu Hasegawa and Tsutomu Iwamoto : 歯科治療恐怖症患者に応用した心理検査結果の経時的変化について, Journal of the Japanese Society for Disability and Oral Health, 34, 3, 375, Sep. 2013.
50.
赤澤 友基, 帖佐 直行, 吉村 善隆, Takamasa Kitamura, Yuri Fujihara, 浅川 剛吉, 石崎 明 and Tomokazu Hasegawa : Establishment of immortalized dental pulp cell lines derived from human deciduous teeth, The Japanese Journal of Pediatric Dentistry, 51, 2, 303, Apr. 2013.
Hiroshi Nakagawa, Aimi Yamamoto, Yukiko Kori, 赤澤 友基, Takamasa Kitamura, Tomokazu Hasegawa and Tsutomu Iwamoto : 歯科治療恐怖症患者に応用した心理検査結果の経時的変化について, Journal of the Japanese Society for Disability and Oral Health, 34, 3, 375, 2013.
Stromal cell-derived factor1 expression on periodontal ligament cells derived from Down syndrom (Project/Area Number: 20K10235 )
Effect of CCL11 derived from deciduous tooth on pulp and root resorption (Project/Area Number: 20K10205 )
Cell fate determination of mesenchymal stem cells by extracellular mechanical stress (Project/Area Number: 17H04414 )
homeostatic regulation and regeneration of periodontal ligament tissues (Project/Area Number: 16K11804 )
Development of the periodontal therapy by the stromal cell derived factor -Analysis of periodontal ligament cells derived from Down's syndrome - (Project/Area Number: 16K11812 )
Elucidation of matrix vesicles secretion mechanism and application to calcification (Project/Area Number: 15K11368 )
role of preodontoblast in tooth development (Project/Area Number: 26293435 )
Study of periodontal ligament tissue function about maintaining homeostasis and regeneration in periodontal tissues, and development of new regeneration therapy in periodontal tissues. (Project/Area Number: 25463182 )
Establishment of periodontal ligament (PDL) cells derived from deciduous teeth and periodontal tissue regeneration with PDL cells. (Project/Area Number: 22592296 )
Analysis of pathological and/or unphysiological root resorption mechanism focused on the two types of osteoclasts (Project/Area Number: 22592274 )
Analysis of periodontal ligament cells derived from permanent teeth and primary teeth on tooth root resorption. (Project/Area Number: 18592239 )
The analysis of the network transcription regulation by the IKK・r in the tooth differentiation and development. (Project/Area Number: 17592125 )
RNAi-induced change in the expression of estrogen receptor α in the median preoptic nuclei of mother rats and its effects on the behavior of their offsprings. (Project/Area Number: 17390555 )
Search for the bioactive molecules implicated in the regulation of human periodontal ligament cell functions (Project/Area Number: 09470464 )
Establishment of Human Fibroblasts deribed from Dental Pulp Tissues of Primary Teeth and Permanent Teeth, and Development of Systems of Cytotoxicity Tests for Dental Materials (Project/Area Number: 08672378 )