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Hiroko Okawa

Section

Graduate School of Dentistry

Job title

Assistant Professor

Degree

P.h.D. (Osaka University)

researchmap

https://researchmap.jp/h-okawa

J-GLOBAL ID

201901019710207338

e-Rad No.

00781296

Research History 6

2024/07 - Present

東北大学プロミネントリサーチフェロー
2020/04 - Present

東北大学大学院歯学研究科　分子・再生歯科補綴学分野　助教
2018/03 - 2020/03

University of California, Los Angeles
2018/03 - 2020/03

Tohoku University　Graduate School of Dentistry
2016/04 - 2018/02

東北大学病院　医員
2011/04 - 2012/03

Osaka University　Dental Hospital

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Education 2

Osaka University　Graduate School of Dentistry

2012/04 - 2016/03
Okayama University　Dental School

2005/04 - 2011/03

Professional Memberships 4

JAPANESE SOCIETY OF ORAL IMPLANTOLOGY
THE JAPANESE SOCIETY FOR REGENERATIVE MEDICINE
International Association for Dental Research (IADR)
JAPAN PROSTHODONTIC SOCIETY

Research Interests 3

歯科補綴学
人工多能性幹細胞
再生医療

Research Areas 2

Life sciences / Prosthodontics /
Life sciences / Regenerative dentistry and dental engineering /

Awards 5

若手リーダー研究者海外派遣プログラム

2018/03　東北大学
歯科医学を中心とした総合的な研究を推進する集い優秀発表賞

2016　日本歯科医学会
優秀海外発表大学院生奨励賞

2015　大阪大学歯学会
デンツプライ賞

2015　日本口腔インプラント学会
Arthur R. Frechette 若手補綴学研究者賞第1位

2014/06　国際歯科研究学会（IADR）

Papers 27

Spheroid size-induced apoptosis enhances osteogenic differentiation of iPS cells Peer-reviewed

Hideto Tatsumi, Hiroko Okawa, Naruephorn Vinaikosol, Akihito Moribayashi, Hiroki Kayashima, Hirofumi Yatani, Hiroshi Egusa

Journal of Bioscience and Bioengineering　2025/12
Publisher: Elsevier BV
DOI： 10.1016/j.jbiosc.2025.11.010 　

ISSN： 1389-1723
Shaking culture attenuates circadian rhythms in induced pluripotent stem cells during osteogenic differentiation through the TEAD-Fbxl3-CRY axis Peer-reviewed

Yunyu Fu, Hiroko Okawa, Naruephorn Vinaikosol, Satomi Mori, Phoonsuk Limraksasin, Praphawi Nattasit, Yu Tahara, Hiroshi Egusa

Cell Death Discovery　11　(1)　2025/05/24
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41420-025-02533-6 　

eISSN： 2058-7716

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Abstract Circadian rhythms, which synchronize cellular and organismal activities with the Earth’s 24-hour light-dark cycle, are controlled by clock genes. These genes not only regulate metabolic and physiological processes but also influence osteogenesis. Despite extensive research on the genetic control of circadian rhythms, little is known about the mechanisms by which mechanical factors in the extracellular environment affect these rhythms during the osteogenic differentiation of induced pluripotent stem cells (iPSCs). Shaking culture, which promotes the formation of three-dimensional organoid-like constructs from iPSC embryoid bodies (iPSC-EBs), introduces distinct biomechanical forces compared with static adherent culture. This raises the question of how these forces affect the circadian gene expression during osteogenic differentiation. In this study, we investigated the effects of shaking cultures on the circadian rhythm of key clock genes (Clock, Bmal1, and Npas2) in iPSC-EBs. In the adherent culture, iPSC-EBs displayed rhythmic oscillations of the clock genes, which were attenuated in the shaking culture. RNA-seq analysis revealed that the yes-associated protein (YAP)-transcriptional enhanced associate domain (TEAD) transcriptional cascade was activated in the shaking culture. Further investigations using assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation assays identified Fbxl3 as a direct target of this transcriptional cascade. Fbxl3 upregulation in the shaking culture enhanced the degradation of CRY proteins, which are essential components of the circadian feedback loop, thereby suppressing clock gene oscillations. In addition, treatment with verteporfin, a YAP-TEAD inhibitor, restored circadian gene oscillations and increased the expression of osteogenic markers in shaking culture. These findings highlight a novel mechanistic link between biomechanical cues and circadian regulation and offer potential insights for optimizing tissue engineering strategies in regenerative medicine.
Induced pluripotent stem cell-derived neural stem cells promote bone formation in mice with calvarial defects

Yukari Shiwaku, Hiroko Okawa, Ikuro Suzuki, Susumu Sakai, Hiroshi Egusa, Osamu Suzuki

Acta Biomaterialia　188　93-102　2024/10
Publisher: Elsevier BV
DOI： 10.1016/j.actbio.2024.08.054 　

ISSN： 1742-7061
Effect of circadian clock disruption on type 2 diabetes. International-journal

Hong Thuan Tran, Takeru Kondo, Amal Ashry, Yunyu Fu, Hiroko Okawa, Chenphop Sawangmake, Hiroshi Egusa

Frontiers in physiology　15　1435848-1435848　2024

DOI： 10.3389/fphys.2024.1435848 　

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INTRODUCTION: Type 2 diabetes (T2D) is the predominant form of diabetes mellitus and is among the leading causes of death with an increasing prevalence worldwide. However, the pathological mechanism underlying T2D remains complex and unclear. An increasing number of studies have suggested an association between circadian clock disruption and high T2D prevalence. METHOD: This review explores the physiological and genetic evidence underlying T2D symptoms associated with circadian clock disturbances, including insulin secretion and glucose metabolism. RESULTS AND DISCUSSION: Notably, circadian clock disruption reduces insulin secretion and insulin sensitivity and negatively affects glucose homeostasis. The circadian clock regulates the hypothalamic-pituitary-adrenal axis, an important factor that regulates glucose metabolism and influences T2D progression. Therefore, circadian clock regulation is an attractive, novel therapeutic approach for T2D, and various circadian clock stabilizers play therapeutic roles in T2D. Lastly, this review suggests novel therapeutic and preventive approaches using circadian clock regulators for T2D.
Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells Peer-reviewed

Jeeranan Manokawinchoke, Phoonsuk Limraksasin, Hiroko Okawa, Prasit Pavasant, Hiroshi Egusa, Thanaphum Osathanon

International Journal of Oral Science　14　(1)　2022/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41368-021-00151-3 　

ISSN： 1674-2818

eISSN： 2049-3169

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Abstract In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.
Oral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin K in mice International-journal Peer-reviewed

Takeru Kondo, Hiroko Okawa, Akishige Hokugo, Bhumika Shokeen, Oskar Sundberg, Yiying Zheng, Charles E. McKenna, Renate Lux, Ichiro Nishimura

Communications Biology　5　(1)　962-962　2022/09/14
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s42003-022-03896-7 　

eISSN： 2399-3642

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Abstract Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk. The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis.
Fluorescent risedronate analogue 800CW-pRIS improves tooth extraction-associated abnormal wound healing in zoledronate-treated mice International-journal Peer-reviewed

Hiroko Okawa, Takeru Kondo, Akishige Hokugo, Philip Cherian, Oskar Sundberg, Jesus J. Campagna, Boris A. Kashemirov, Varghese John, Shuting Sun, Frank H. Ebetino, Charles E. McKenna, Ichiro Nishimura

Communications Medicine　2　(1)　112-112　2022/09/05
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s43856-022-00172-x 　

eISSN： 2730-664X

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Abstract Background Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a rare but serious side effect of nitrogen-containing bisphosphonate drugs (N-BPs) frequently prescribed to reduce skeletal-related events in bone malignancies and osteoporosis. BRONJ is associated with abnormal oral wound healing after dentoalveolar surgery and tooth extraction. We previously found that N-BP chemisorbed to bone mineral hydroxyapatite was dissociated by secondary applied N-BP. This study investigated the effect of the surface equilibrium-based removal of N-BP from jawbone on tooth extraction wound healing of zoledronate (ZOL)-treated mice. Methods A pharmacologically inactive N-BP derivative (the 4-pyridyl isomer of risedronate equipped with a near-infrared 800CW fluorescent imaging dye, 800CW-pRIS) was designed and synthesized. 800CW-pRIS was intra-orally injected or topically applied in a deformable nano-scale vesicle formulation (DNV) to the palatal tissue of mice pretreated with ZOL, a potent N-BP. The female C56BL6/J mice were subjected to maxillary molar extraction and oral wound healing was compared for 800CW-pRIS/ZOL, ZOL and untreated control groups. Results 800CW-pRIS is confirmed to be inactive in inhibiting prenylation in cultured osteoclasts while retaining high affinity for hydroxyapatite. ZOL-injected mice exhibit delayed tooth extraction wound healing with osteonecrosis relative to the untreated controls. 800CW-pRIS applied topically to the jaw one week before tooth extraction significantly reduces gingival oral barrier inflammation, improves extraction socket bone regeneration, and prevents development of osteonecrosis in ZOL-injected mice. Conclusions Topical pre-treatment with 800CW-RIS in DNV is a promising approach to prevent the complication of abnormal oral wound healing associated with BRONJ while retaining the anti-resorptive benefit of legacy N-BP in appendicular or vertebrate bones.
Mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) revealed by targeted removal of legacy bisphosphonate from jawbone using equilibrium competing inert hydroxymethylene diphosphonate International-journal Peer-reviewed

Hiroko Okawa, Takeru Kondo, Akishige Hokugo, Philip Cherian, Jesus J Campagna, Nicholas A Lentini, Eric C Sung, Samantha Chiang, Yi-Ling Lin, Frank H Ebetino, Varghese John, Shuting Sun, Charles E McKenna, Ichiro Nishimura

eLife　11　2022/08/26
Publisher: eLife Sciences Publications, Ltd
DOI： 10.7554/elife.76207 　

eISSN： 2050-084X

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Bisphosphonate-related osteonecrosis of the jaw (BRONJ) presents as a morbid jawbone lesion in patients exposed to a nitrogen-containing bisphosphonate (N-BP). Although it is rare, BRONJ has caused apprehension among patients and healthcare providers and decreased acceptance of this anti-resorptive drug class to treat osteoporosis and metastatic osteolysis. We report here a novel method to elucidate the pathological mechanism of BRONJ by the selective removal of legacy N-BP from the jawbone using an intra-oral application of hydroxymethylene diphosphonate (HMDP) formulated in liposome-based deformable nanoscale vesicles (DNV). After maxillary tooth extraction, zoledronate-treated mice developed delayed gingival wound closure, delayed tooth extraction socket healing and increased jawbone osteonecrosis consistent with human BRONJ lesion. Single cell RNA sequencing of mouse gingival cells revealed oral barrier immune dysregulation and unresolved pro-inflammatory reaction. HMDP-DNV topical applications to nascent mouse BRONJ lesions resulted in accelerated gingival wound closure and bone socket healing as well as attenuation of osteonecrosis development. The gingival single cell RNA sequencing demonstrated resolution of chronic inflammation by increased anti-inflammatory signature gene expression of lymphocytes and myeloid-derived suppressor cells. This study suggests that BRONJ pathology is related to N-BP levels in jawbones and demonstrates the potential of HMDP-DNV as an effective BRONJ therapy.
Application of Shear Stress for Enhanced Osteogenic Differentiation of Mouse Induced Pluripotent Stem Cells Peer-reviewed

Phoonsuk Limraksasin, Praphawi Nattasit, Jeeranan Manokawinchoke, Watcharaphol Tiskratok, Naruephorn Vinaikosol, Hiroko Okawa, Chalida Nakalekha Limjeerajarus, Nuttapol Limjeerajarus, Prasit Pavasant, Thanaphum Osathanon, Hiroshi Egusa

2022/07/25
Publisher: Research Square Platform LLC
DOI： 10.21203/rs.3.rs-1815026/v1 　

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Abstract The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.
Rapid and Efficient Generation of Cartilage Pellets from Mouse Induced Pluripotent Stem Cells by Transcriptional Activation of BMP-4 with Shaking Culture. Peer-reviewed

Zhang M, Niibe K, Kondo T, Limraksasin P, Okawa H, Miao X, Kamano Y, Yamada M, Jiang X, Egusa H

J Tissue Eng.　13　(20417314221114616)　2022/07

DOI： 10.1177/20417314221114616 　
Cell-Based Double-Screening Method to Identify a Reliable Candidate for Osteogenesis-Targeting Compounds Peer-reviewed

Sho Fukuyasu, Hiroki Kayashima, Akihito Moribayashi, Shu Matsuoka, Atsuhiro Nagasaki, Hiroko Okawa, Hirofumi Yatani, Makio Saeki, Hiroshi Egusa

Biomedicines　10　(2)　426-426　2022/02/11
Publisher: MDPI AG
DOI： 10.3390/biomedicines10020426 　

eISSN： 2227-9059

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Small-molecule compounds strongly affecting osteogenesis can form the basis of effective therapeutic strategies in bone regenerative medicine. A cell-based high-throughput screening system might be a powerful tool for identifying osteoblast-targeting candidates; however, this approach is generally limited with using only one molecule as a cell-based sensor that does not always reflect the activation of the osteogenic phenotype. In the present study, we used the MC3T3-E1 cell line stably transfected with the green fluorescent protein (GFP) reporter gene driven by a fragment of type I collagen promoter (Col-1a1GFP-MC3T3-E1) to evaluate a double-screening system to identify osteogenic inducible compounds using a combination of a cell-based reporter assay and detection of alkaline phosphatase (ALP) activity. Col-1a1GFP-MC3T3-E1 cells were cultured in an osteogenic induction medium after library screening of 1280 pharmacologically active compounds (Lopack1280). After 7 days, GFP fluorescence was measured using a microplate reader. After 14 days of osteogenic induction, the cells were stained with ALP. Library screening using the Col-1a1/GFP reporter and ALP staining assay detected three candidates with significant osteogenic induction ability. Furthermore, leflunomide, one of the three detected candidates, significantly promoted new bone formation in vivo. Therefore, this double-screening method could identify candidates for osteogenesis-targeting compounds more reliably than conventional methods.
Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing International-journal Peer-reviewed

Yoichiro Shibuya, Akishige Hokugo, Hiroko Okawa, Takeru Kondo, Daniel Khalil, Lixin Wang, Yvonne Roca, Adam Clements, Hodaka Sasaki, Ella Berry, Ichiro Nishimura, Reza Jarrahy

eLife　11　2022/01/18
Publisher: eLife Sciences Publications, Ltd
DOI： 10.7554/elife.71074 　

eISSN： 2050-084X

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Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (<italic>Npas2</italic>)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates <italic>Npas2</italic> activity. The hit compound (Dwn1) suppressed circadian <italic>Npas2</italic> expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgfβ1, and α-smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.
Cyclic pressure-induced cytokines from gingival fibroblasts stimulate osteoclast activity: Clinical implications for alveolar bone loss in denture wearers. Peer-reviewed

Akashi Y, Nagasaki A, Okawa H, Matsumoto T, Kondo T, Yatani H, Nishimura I, Egusa H

Journal of prosthodontic research　2022

DOI： 10.2186/jpr.JPR_D_21_00238 　

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PURPOSE: The involvement of oral mucosa cells in mechanical stress-induced bone resorption is unclear. The aim of this study was to investigate the effects of cyclic pressure-induced cytokines from oral mucosal cells (human gingival fibroblasts: hGFs) on osteoclast activity in vitro. METHODS: Cyclic pressure at 50 kPa, which represents high physiologic occlusal force of dentures on the molar area, was applied to hGFs. NFAT-reporter stable RAW264.7 preosteoclasts (NFAT/Luc-RAW cells) were cultured in conditioned medium collected from hGF cultures under cyclic pressure or static conditions. NFAT activity and osteoclast formation were determined by luciferase reporter assay and TRAP staining, respectively. Cyclic pressure-induced cytokines in hGF culture were detected by ELISA, real-time RT-PCR, and cytokine array analyses. RESULTS: Conditioned media from hGFs treated with 48 hours of cyclic pressure significantly induced NFAT activity and increased multinucleated osteoclast formation. Furthermore, the cyclic pressure significantly increased the bone resorption activity of RAW264.7 cells. Cyclic pressure significantly increased the expression of major inflammatory cytokines including IL-1β/IL-1β, IL-6/IL-6, IL-8/IL-8 and MCP-1/CCL2 in hGFs compared to hGFs cultured under static conditions, and it suppressed osteoprotegerin (OPG/OPG) expression. A cytokine array detected 12 cyclic pressure-induced candidates. Among them, IL-8, decorin, MCP-1 and ferritin increased, whereas IL-28A and PDGF-BB decreased, NFAT activation of NFAT/Luc-RAW cells. CONCLUSION: These results suggest that cyclic pressure-induced cytokines from hGFs promote osteoclastogenesis, possibly including up-regulation of IL-1β, IL-6, IL-8 and MCP-1, and down-regulation of OPG. These findings introduce the possible involvement of GFs in mechanical stress-induced alveolar ridge resorption, such as in denture wearers.
Point-of-care antimicrobial coating protects orthopaedic implants from bacterial challenge International-journal Peer-reviewed

Weixian Xi, Vishal Hegde, Stephen D. Zoller, Howard Y. Park, Christopher M. Hart, Takeru Kondo, Christopher D. Hamad, Yan Hu, Amanda H. Loftin, Daniel O. Johansen, Zachary Burke, Samuel Clarkson, Chad Ishmael, Kellyn Hori, Zeinab Mamouei, Hiroko Okawa, Ichiro Nishimura, Nicholas M. Bernthal, Tatiana Segura

Nature Communications　12　(1)　5473-5473　2021/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41467-021-25383-z 　

eISSN： 2041-1723

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<title>Abstract</title>Implant related infections are the most common cause of joint arthroplasty failure, requiring revision surgeries and a new implant, resulting in a cost of $8.6 billion annually. To address this problem, we created a class of coating technology that is applied in the operating room, in a procedure that takes less than 10 min, and can incorporate any desired antibiotic. Our coating technology uses an in situ coupling reaction of branched poly(ethylene glycol) and poly(allyl mercaptan) (PEG-PAM) polymers to generate an amphiphilic polymeric coating. We show in vivo efficacy in preventing implant infection in both post-arthroplasty infection and post-spinal surgery infection mouse models. Our technology displays efficacy with or without systemic antibiotics, the standard of care. Our coating technology is applied in a clinically relevant time frame, does not require modification of implant manufacturing process, and does not change the implant shelf life.
Shaking culture enhances chondrogenic differentiation of mouse induced pluripotent stem cell constructs Peer-reviewed

Phoonsuk Limraksasin, Yukihiro Kosaka, Maolin Zhang, Naohiro Horie, Takeru Kondo, Hiroko Okawa, Masahiro Yamada, Hiroshi Egusa

Scientific Reports　10　(1)　2020/12
Publisher: Springer Science and Business Media LLC
DOI： 10.1038/s41598-020-72038-y 　

eISSN： 2045-2322
Size-Optimized Microspace Culture Facilitates Differentiation of Mouse Induced Pluripotent Stem Cells into Osteoid-Rich Bone Constructs International-journal Peer-reviewed

Phoonsuk Limraksasin, Hiroko Okawa, Maolin Zhang, Takeru Kondo, Thanaphum Osathanon, Prasit Pavasant, Hiroshi Egusa

Stem Cells International　2020　1-14　2020/05/14
Publisher: Hindawi Limited
DOI： 10.1155/2020/7082679 　

ISSN： 1687-966X

eISSN： 1687-9678

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Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue <italic>in vitro</italic>. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 <italic>μ</italic>m (Elp400), 500/700 <italic>μ</italic>m (Elp500), and 900/700 <italic>μ</italic>m (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of <italic>Runx2</italic>, <italic>Osterix</italic>, <italic>Collagen 1a1</italic>, <italic>Osteocalcin</italic>, <italic>Bone sialoprotein</italic>, and <italic>Osteopontin</italic> compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat’s pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.
In vitro fabrication of hybrid bone/cartilage complex using mouse induced pluripotent stem cells Peer-reviewed

Limraksasin P, Kondo T, Zhang M, Okawa H, Osathanon T, Pavasant P, Egusa H

Int J Mol Sci　21　(2)　in press-581　2020/01
Publisher: MDPI AG
DOI： 10.3390/ijms21020581 　

eISSN： 1422-0067

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Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.
Implications of the circadian clock in implant dentistry Peer-reviewed

Okawa H, Egusa H, Nishimura I

Dental Materials Journal　2019/11
Rescue bisphosphonate treatment of alveolar bone improves extraction socket healing and reduces osteonecrosis in zoledronate-treated mice. Peer-reviewed

Hokugo A, Kanayama K, Sun S, Morinaga K, Sun Y, Wu Q, Sasaki H, Okawa H, Evans C, Ebetino FH, Lundy MW, Sadrerafi K, McKenna CE, Nishimura I

Bone　123　115-128　2019/06

DOI： 10.1016/j.bone.2019.03.027 　

ISSN： 8756-3282
Neuronal PAS Domain 2 (Npas2)-Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction. Peer-reviewed

Sasaki H, Hokugo A, Wang L, Morinaga K, Ngo JT, Okawa H, Nishimura I

Anatomical record (Hoboken, N.J. : 2007)　2019/03

DOI： 10.1002/ar.24109 　

ISSN： 1932-8486
Neuronal PAS domain 2 (Npas2) facilitated osseointegration of titanium implant with rough surface through a neuroskeletal mechanism. International-journal Peer-reviewed

Kenzo Morinaga, Hodaka Sasaki, Sil Park, Akishige Hokugo, Hiroko Okawa, Yu Tahara, Christopher S Colwell, Ichiro Nishimura

Biomaterials　192　62-74　2019/02

DOI： 10.1016/j.biomaterials.2018.11.003 　

ISSN： 0142-9612

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Titanium (Ti) biomaterials have been applied to a wide range of implantable medical devices. When placed in bone marrow, Ti-biomaterials integrate to the surrounding bone tissue by mechanisms that are not fully understood. We have previously identified an unexpected upregulation of circadian clock molecule neuronal PAS domain 2 (Npas2) in successfully integrated implant with a rough surface. This study aimed to elucidate the molecular mechanism of osseointegration through determining the role of Npas2. Human bone marrow stromal cells (BMSC) that were cultured on a Ti disc with SLA surface exhibited increased NPAS2 expression compared to BMSC cultured on a machined surface. A mouse model was developed in which miniature Ti implants were surgically placed into femur bone marrow. The implant push-out test and bone-to-implant contact measurements demonstrated the establishment of osseointegration in 3 weeks. By contrast, in Npas2 functional knockout (KO) mice, the implant push-out value measured for SLA surface Ti implant was significantly decreased. Npas2 KO mice demonstrated normal femur bone structure surrounding the Ti implant; however, the recovered implants revealed abnormal remnant mineralized tissue, which lacked dense collagen architecture typically found on recovered implants from wild type mice. To explore the mechanisms leading to the induced Npas2 expression, an unbiased chemical genetics analysis was conducted using mouse BMSC carrying an Npas2-reporter gene for high throughput screening of Library of Pharmacologically Active Compounds. Npas2 modulating compounds were found clustered in regulatory networks of the α2-adrenergic receptor and its downstream cAMP/CREB signaling pathway. Mouse primary BMSC exposed to SLA Ti disc significantly increased the expression of α2-adrenergic receptors, but the expression of β2-adrenergic receptor was unaffected. Our data provides the first evidence that peripheral clock gene component Npas2 plays a role in facilitating the enhanced osseointegration through neuroskeletal regulatory pathways induced by BMSC in contact with rough surface Ti implant.
Binding of PICK1 PDZ domain with calcineurin B regulates osteoclast differentiation Peer-reviewed

Yuya Kamano, Jun Watanabe, Tsutomu Iida, Takeru Kondo, Hiroko Okawa, Hirofumi Yatani, Makio Saeki, Hiroshi Egusa

Biochemical and Biophysical Research Communications　496　(1)　83-88　2018/01/29
Publisher: Elsevier B.V.
DOI： 10.1016/j.bbrc.2017.12.173 　

ISSN： 1090-2104 0006-291X
Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells Peer-reviewed

G. Yu, H. Okawa, K. Okita, Y. Kamano, F. Wang, M. Saeki, H. Yatani, H. Egusa

JOURNAL OF DENTAL RESEARCH　95　(1)　110-118　2016/01

DOI： 10.1177/0022034515611602 　

ISSN： 0022-0345

eISSN： 1544-0591
Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells Peer-reviewed

Hiroko Okawa, Hiroki Kayashima, Jun-Ichi Sasaki, Jiro Miura, Yuya Kamano, Yukihiro Kosaka, Satoshi Imazato, Hirofumi Yatani, Takuya Matsumoto, Hiroshi Egusa

STEM CELLS INTERNATIONAL　2016　6240794　2016

DOI： 10.1155/2016/6240794 　

ISSN： 1687-966X

eISSN： 1687-9678
Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin Peer-reviewed

Fangfang Wang, Hiroko Okawa, Yuya Kamano, Kunimichi Niibe, Hiroki Kayashima, Thanaphum Osathanon, Prasit Pavasant, Makio Saeki, Hirofumi Yatani, Hiroshi Egusa

PLOS ONE　10　(12)　e0145677　2015/12

DOI： 10.1371/journal.pone.0145677 　

ISSN： 1932-6203
Feeder cell sources and feeder-free methods for human ips cell culture Invited

Guannan Yu, Fangfang Wang, Hiroko Okawa, Hirofumi Yatani

Interface Oral Health Science 2014: Innovative Research on Biosis-Abiosis Intelligent Interface　2014　145-159　2015/01/01
Publisher: Springer Japan
DOI： 10.1007/978-4-431-55192-8_12 　
Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro Peer-reviewed

Hiroshi Egusa, Hiroki Kayashima, Jiro Miura, Shinya Uraguchi, Fangfang Wang, Hiroko Okawa, Jun-Ichi Sasaki, Makio Saeki, Takuya Matsumoto, Hirofumi Yatani

STEM CELLS AND DEVELOPMENT　23　(18)　2156-2169　2014/09

DOI： 10.1089/scd.2013.0344 　

ISSN： 1547-3287

eISSN： 1557-8534

Show all ︎Show first 5

Misc. 3

ヒトiPS細胞に低酸素培養を応用した再生医療用骨組織の作製

大川博子, 江草宏

日本歯科医学会誌　36　60-60　2017/03
Publisher: 日本歯科医学会
ISSN： 0286-164X
iPS細胞を用いた唾液腺再生への試み

小野瞳, 尾花綾, 宇佐美悠, 江草宏, 大川博子, 山城隆, 野原幹司, 阪井丘芳

日本口腔科学会雑誌　63　(4)　393-393　2014/09
Publisher: (NPO)日本口腔科学会
ISSN： 0029-0297

eISSN： 2185-0461
歯肉を用いた医療応用に安全なiPS細胞の開発新たな再生歯科医療技術の創成に向けて

江草宏, 于冠男, 鎌野優弥, 大川博子, 山本治毅, 矢谷博文

日本歯科医学会誌　33　54-58　2014/03
Publisher: 日本歯科医学会
ISSN： 0286-164X

Presentations 4

OFS, Novel Sentinel Imaging Probes for Early Diagnosis of Periodontitis

Hiroko Okawa, Akishige Hokugo, Lixin Wang, Daniel Khalil, Shota Homma, Eric Richard, Yiying Zheng, Boris Kashemirov, Reza Jarrahy, Charles McKenna, Ichiro Nishimura

米国骨代謝学会（ASBMR)　2019/09
Enhanced osteogenesis of mouse BMSC by Npas2 down-regulation

Hiroko Okawa, Akishige Hokugo, Ichiro Nishimura

国際歯科研究学会（IADR）　2019/06
iPS細胞は歯科補綴学にどのように生かされるのか？

黄地健仁, 新部邦透, 大川博子, 帆足有理恵

第126回日本補綴歯科学会　2017/07
Vertical Bone Augmentation Using iPS Cell Constructs with Simvastatin

Okawa H, Kayashima H, Yamamoto H, Yatani H, Egusa H

国際歯科研究学会（IADR）　2014/06

Industrial Property Rights 1

ヒトiPS細胞を用いた骨芽細胞塊の作製法

江草宏, 大川博子

Property Type: Patent

Research Projects 5

義歯装着が骨吸収抑制薬関連顎骨壊死に及ぼす影響の解明

大川博子, 山本雅哉, 江草宏

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 基盤研究(C)

Institution: 東北大学

2024/04/01 - 2027/03/31
リンパ行性の幹細胞/薬剤送達を利用した革新的顎骨壊死治療法の創成

江草宏, 大川博子, 小玉哲也, 新部邦透

Offer Organization: 日本学術振興会

System: 科学研究費助成事業

Category: 挑戦的研究(萌芽)

Institution: 東北大学

2023/06/30 - 2025/03/31

More details Close

近年、間葉系幹細胞（MSC）の免疫機能調節能力に注目が集まり、MSCの静脈投与は薬剤関連顎骨壊死（MRONJ）等の難治性炎症性疾患に対して免疫を制御する画期的な治療法として期待されている。しかし、静脈血に投与した細胞のほとんどは肺に捕捉されるため、副作用や患部に到達し難いという根本的な課題がある。本研究では、「リンパ行性薬剤送達法」に着目し、MRONJに対してリンパ節内に投与したMSCをリンパ行性に患部に送達させる治療アプローチを探索し、その作用機序を細胞遊走および免疫応答の観点から理解することを目的とする。初年度は、予備実験としてMRL/MpJmsSlc-lpr/lprマウスの大腿骨骨髄より分離培養したMSCを、全身のリンパ節が生まれつき腫脹しておりリンパ節に細胞の投与が容易なMXH51マウスの腸骨下リンパ節内および比較対象群の尾静脈内に投与する実験系を確立した。また、投与する細胞数がリンパ節組織におけるCD4、CD８および炎症性サイトカインの発現に及ぼす影響を検討し、至適投与細胞数を決定した。投与した腸骨下リンパ節と伝達先の下流にある固有腋窩リンパ節におけるこれら分子の遺伝子発現について検討した結果、下流のリンパ節においてより著明な免疫応答を認めた。また、肺の組織切片観察の結果、尾静脈内投与群では投与した細胞に起因するとみられる炎症性細胞浸潤を認めたが、リンパ節内投与群ではそのような所見を認めなかったことから、リンパ行性薬剤送達法は静脈投与よりも副作用の少ない細胞投与法である可能性が示唆された。今後、免疫応答の詳細と共に顎骨壊死（ONJ）発症モデルを用いた検討を行う予定である。
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Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists

Category: Grant-in-Aid for Early-Career Scientists

Institution: Tohoku University

2022/04 - 2024/03
Development of a method for the endochondral alveolar bone tissue engineering using iPS cells

Egusa Hiroshi, SAITO Masahiro, FUKUMOTO Satoshi, OKAWA Hiroko, KOSAKA Yukihiro

Offer Organization: Japan Society for the Promotion of Science

System: Grants-in-Aid for Scientific Research

Category: Grant-in-Aid for Challenging Exploratory Research

Institution: Tohoku University

2016/04/01 - 2018/03/31

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A strategy of induced pluripotent stem (iPS) cell-based alveolar bone regeneration is expected to improve treatment outcome in prosthetic treatments. We focused on possible advantage in endochondral bone regeneration in the large bone defect. The aim of this study was to develop a method to fabricate bone/cartilage-like cell constructs using iPS cells. We found that combination use of chondrogenic and osteogenic induction factors could guide mouse iPS cell embryoid bodies into hybrid bone/cartilage-like cell constructs on the manipulation of a ratio in degrees of chondrocyte/osteoblast differentiation.
ヒトiPS細胞に低酸素培養を応用した三次元骨様組織の作製およびその骨組織再生効果 Competitive

大川博子

Offer Organization: 日本学術振興会

System: 研究活動スタート支援

2016/09 - 2018/03