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1
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Okuda K, Kaori K, Kawauchi A, Miyu I, Yomogida K. An oscillating magnetic field suppresses ice-crystal growth during rapid freezing of muscle tissue of mice. J Biochem 2024; 175:245-252. [PMID: 37948636 DOI: 10.1093/jb/mvad087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 11/02/2023] [Indexed: 11/12/2023] Open
Abstract
Regenerative medicine would benefit from a safe and efficient cryopreservation method to prevent the structural disruption caused by ice-crystal formation in cells and tissue. Various attempts have been made to overcome this problem, one of which is the use of an oscillating magnetic field (OMF). However, the underlying mechanism is unclear. In this study, to evaluate the effect of an OMF on ice-crystal formation in the leg muscles of mice, we used to use the frozen-section method with a slower freezing rate than is, usual which resulted in ice crystals forming in the tissue. We assessed the mean size and number per unit area of intracellular ice holes in sections of muscle tissue, with and without OMF. Ice-crystal growth was reduced in frozen tissue subjected to OMF. Furthermore, we evaluated the structure and function of proteins in frozen tissue subjected to OMF by immunostaining using an anti-dystrophin antibody and by enzymatic histochemistry for NADH-TR and myosin ATPase. The results imply that the ability of OMF to suppress ice-crystal growth might be related to their stabilization of bound water in biomolecules during freezing.
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Affiliation(s)
- Kana Okuda
- Department of Innovative Food Science, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
- Abi Inc., Ohtakanomori-higashi 1-12-1 270-0138, Nagareyama, Japan
| | - Kunitani Kaori
- Department of Innovative Food Science, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
| | - Aiko Kawauchi
- Department of Innovative Food Science, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
- Abi Inc., Ohtakanomori-higashi 1-12-1 270-0138, Nagareyama, Japan
| | - Ishii Miyu
- Department of Innovative Food Science, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
- Abi Inc., Ohtakanomori-higashi 1-12-1 270-0138, Nagareyama, Japan
| | - Kentaro Yomogida
- Department of Innovative Food Science, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
- Institute for Bioscience, Mukogawa Women's University, Ikebiraki-cho 6-46, 663-8558 Nishinomiya, Japan
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2
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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3
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Zhu Y, Wei SM, Yan KX, Gu YX, Lai HC, Qiao SC. Bovine-Derived Xenografts Immobilized With Cryopreserved Stem Cells From Human Adipose and Dental Pulp Tissues Promote Bone Regeneration: A Radiographic and Histological Study. Front Bioeng Biotechnol 2021; 9:646690. [PMID: 33912548 PMCID: PMC8075412 DOI: 10.3389/fbioe.2021.646690] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2020] [Accepted: 03/22/2021] [Indexed: 01/09/2023] Open
Abstract
Adipose tissue-derived stem cells (ADSCs) and dental pulp stem cells (DPSCs) have become promising sources for bone tissue engineering. Our study aimed at evaluating bone regeneration potential of cryopreserved ADSCs and DPSCs combined with bovine-derived xenografts with 10% porcine collagen. In vitro studies revealed that although DPSCs had higher proliferative abilities, ADSCs exhibited greater mineral depositions and higher osteogenic-related gene expression, indicating better osteogenic differentiation potential of ADSCs. After applying cryopreserved ADSCs and DPSCs in a critical-sized calvarial defect model, both cryopreserved mesenchymal stem cells significantly improved bone volume density and new bone area at 2, 4, and 8 weeks. Furthermore, the combined treatment with ADSCs and xenografts was more efficient in enhancing bone repair processes compared to combined treatment with DPCSs at all-time points. We also evaluated the sequential early bone healing process both histologically and radiographically, confirming a high agreement between these two methods. Based on these results, we propose grafting of the tissue-engineered construct seeded with cryopreserved ADSCs as a useful strategy in accelerating bone healing processes.
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Affiliation(s)
- Yu Zhu
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
| | - Shi-Min Wei
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
| | - Kai-Xiao Yan
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
| | - Ying-Xin Gu
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
| | - Hong-Chang Lai
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
| | - Shi-Chong Qiao
- Department of Implant Dentistry, Shanghai Ninth People's Hospital, College of Stomatology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Jiao Tong University, Shanghai, China
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4
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Izumino J, Kaku M, Yamamoto T, Yashima Y, Kagawa H, Ikeda K, Shimoe S, Tanimoto K. Effects of hyperbaric oxygen treatment on calvarial bone regeneration in young and adult mice. Arch Oral Biol 2020; 117:104828. [DOI: 10.1016/j.archoralbio.2020.104828] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 07/04/2020] [Accepted: 07/07/2020] [Indexed: 12/21/2022]
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5
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Tang J, Zhang H, Tian C, Shao S. Effects of different magnetic fields on the freezing parameters of cherry. J FOOD ENG 2020. [DOI: 10.1016/j.jfoodeng.2020.109949] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
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Hashimoto T, Kazufumi S, Satoru O, Kazuhiko N. Effect of the Cell Alive System on nerve tissue cryopreservation. Cell Tissue Bank 2020; 21:139-149. [PMID: 31912342 DOI: 10.1007/s10561-019-09807-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2019] [Accepted: 12/27/2019] [Indexed: 10/25/2022]
Abstract
Effective cellular cryopreservation while maintaining high cell viability is achieved by preventing intracellular and extracellular ice crystal formation using the Cells Alive System (CAS), a programmed freezer that applies a magnetic field. Here, the optimal temperature settings of the CAS were determined using rat sciatic nerves as a model tissue. Firstly, it was found that Schwann cell survival was increased by pre-cooling the samples in the ice crystal formation zone, increasing the freeze-thaw speed, and freezing-thawing in a magnetic field. Secondly, the setting (intensity and frequency) of the magnetic field at freezing-thawing was changed, and the optimum magnetic field strength was determined by evaluating cell viability. At the set temperature excluding previous studies, the minimum temperature was set to - 50 °C and kept frozen for 15 min, and then thawed immediately. The highest cell viability (27%) was achieved at 0.67 mT (intensity 3 [29.6 V] and frequency setting 10 [60 Hz]). The effects of the freeze-thaw program were assessed using transplanted sciatic nerve tissues removed after 2, 4, and 8 weeks. Anterior tibial muscle wet weight increased at 8 weeks in the control (without freezing) and after freezing-thawing in a magnetic field, compared to that without a magnetic field. Fluorescence staining of the sciatic nerve with anti-S100 antibodies revealed that Schwann cell counts increased at the transplanted site (at 8 weeks) of nerves that were freeze-thawed in a magnetic field. Overall, the CAS prevented ice crystal formation in rat sciatic nerves and could be used to maintain cell viability during cryopreservation.
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Affiliation(s)
- Tomohisa Hashimoto
- Department of Orthopedic Surgery, Dokkyo Medical University Saitama Medical Center, Koshigaya City, Saitama, 343-8555, Japan.
| | - Sano Kazufumi
- Department of Orthopedic Surgery, Dokkyo Medical University Saitama Medical Center, Koshigaya City, Saitama, 343-8555, Japan
| | - Ozeki Satoru
- Department of Orthopedic Surgery, Dokkyo Medical University Saitama Medical Center, Koshigaya City, Saitama, 343-8555, Japan
| | - Nakadate Kazuhiko
- Meiji Pharmaceutical University Pharmaceutical Education Research Center, Kiyose-City, Tokyo, 204-8588, Japan
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Kaur M, Kumar M. An Innovation in Magnetic Field Assisted Freezing of Perishable Fruits and Vegetables: A Review. FOOD REVIEWS INTERNATIONAL 2019. [DOI: 10.1080/87559129.2019.1683746] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Affiliation(s)
- Maninder Kaur
- Department of Processing and Food Engineering, Punjab Agricultural University, Ludhiana, India
| | - Mahesh Kumar
- Department of Processing and Food Engineering, Punjab Agricultural University, Ludhiana, India
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8
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Xia Y, Sun J, Zhao L, Zhang F, Liang XJ, Guo Y, Weir MD, Reynolds MA, Gu N, Xu HHK. Magnetic field and nano-scaffolds with stem cells to enhance bone regeneration. Biomaterials 2018; 183:151-170. [PMID: 30170257 DOI: 10.1016/j.biomaterials.2018.08.040] [Citation(s) in RCA: 171] [Impact Index Per Article: 24.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Revised: 08/10/2018] [Accepted: 08/20/2018] [Indexed: 12/20/2022]
Abstract
Novel strategies utilizing magnetic nanoparticles (MNPs) and magnetic fields are being developed to enhance bone tissue engineering efficacy. This article first reviewed cutting-edge research on the osteogenic enhancements via magnetic fields and MNPs. Then the current developments in magnetic strategies to improve the cells, scaffolds and growth factor deliveries were described. The magnetic-cell strategies included cell labeling, targeting, patterning, and gene modifications. MNPs were incorporated to fabricate magnetic composite scaffolds, as well as to construct delivery systems for growth factors, drugs and gene transfections. The novel methods using magnetic nanoparticles and scaffolds with magnetic fields and stem cells increased the osteogenic differentiation, angiogenesis and bone regeneration by 2-3 folds over those of the controls. The mechanisms of magnetic nanoparticles and scaffolds with magnetic fields and stem cells to enhance bone regeneration were identified as involving the activation of signaling pathways including MAPK, integrin, BMP and NF-κB. Potential clinical applications of magnetic nanoparticles and scaffolds with magnetic fields and stem cells include dental, craniofacial and orthopedic treatments with substantially increased bone repair and regeneration efficacy.
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Affiliation(s)
- Yang Xia
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu 210029, China; Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China; Department of Advanced Oral Sciences & Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Jianfei Sun
- Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China
| | - Liang Zhao
- Department of Advanced Oral Sciences & Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA; Department of Orthopedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
| | - Feimin Zhang
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu 210029, China; Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou, Jiangsu 215123, China
| | - Xing-Jie Liang
- Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China
| | - Yu Guo
- Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu 210029, China
| | - Michael D Weir
- Department of Advanced Oral Sciences & Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Mark A Reynolds
- Department of Advanced Oral Sciences & Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA
| | - Ning Gu
- Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu 210096, China; Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou, Jiangsu 215123, China.
| | - Hockin H K Xu
- Department of Advanced Oral Sciences & Therapeutics, University of Maryland School of Dentistry, Baltimore, MD 21201, USA; Center for Stem Cell Biology and Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA; University of Maryland Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
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9
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Shikata H, Kaku M, Kojima SI, Sumi H, Kojima ST, Yamamoto T, Yashima Y, Kawata T, Tanne K, Tanimoto K. The effect of magnetic field during freezing and thawing of rat bone marrow-derived mesenchymal stem cells. Cryobiology 2016; 73:15-9. [DOI: 10.1016/j.cryobiol.2016.06.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Revised: 06/16/2016] [Accepted: 06/21/2016] [Indexed: 12/22/2022]
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10
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Otero L, Rodríguez AC, Pérez-Mateos M, Sanz PD. Effects of Magnetic Fields on Freezing: Application to Biological Products. Compr Rev Food Sci Food Saf 2016; 15:646-667. [DOI: 10.1111/1541-4337.12202] [Citation(s) in RCA: 84] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2015] [Revised: 02/03/2016] [Accepted: 02/04/2016] [Indexed: 01/11/2023]
Affiliation(s)
- Laura Otero
- Inst. of Food Science, Technology; and Nutrition (ICTAN-CSIC); c/ José Antonio Novais; 10, 28040 Madrid Spain
| | - Antonio C. Rodríguez
- Inst. of Food Science, Technology; and Nutrition (ICTAN-CSIC); c/ José Antonio Novais; 10, 28040 Madrid Spain
| | - Miriam Pérez-Mateos
- Inst. of Food Science, Technology; and Nutrition (ICTAN-CSIC); c/ José Antonio Novais; 10, 28040 Madrid Spain
| | - Pedro D. Sanz
- Inst. of Food Science, Technology; and Nutrition (ICTAN-CSIC); c/ José Antonio Novais; 10, 28040 Madrid Spain
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11
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Nishiyama Y, Iwanami A, Kohyama J, Itakura G, Kawabata S, Sugai K, Nishimura S, Kashiwagi R, Yasutake K, Isoda M, Matsumoto M, Nakamura M, Okano H. Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field. Neurosci Res 2016; 107:20-9. [PMID: 26804710 DOI: 10.1016/j.neures.2015.11.011] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 11/30/2015] [Accepted: 11/30/2015] [Indexed: 02/06/2023]
Abstract
Stem cells represent a potential cellular resource in the development of regenerative medicine approaches to the treatment of pathologies in which specific cells are degenerated or damaged by genetic abnormality, disease, or injury. Securing sufficient supplies of cells suited to the demands of cell transplantation, however, remains challenging, and the establishment of safe and efficient cell banking procedures is an important goal. Cryopreservation allows the storage of stem cells for prolonged time periods while maintaining them in adequate condition for use in clinical settings. Conventional cryopreservation systems include slow-freezing and vitrification both have advantages and disadvantages in terms of cell viability and/or scalability. In the present study, we developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). This system significantly increased cell viability after thawing and had less impact on cellular proliferation and differentiation. We further found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozen ones at the transcriptome level. Given these findings, we suggest that the CAS is useful for hiPSC-NS/PCs banking for clinical uses involving neural disorders and may open new avenues for future regenerative medicine.
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Affiliation(s)
- Yuichiro Nishiyama
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Akio Iwanami
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Jun Kohyama
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Go Itakura
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Soya Kawabata
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Keiko Sugai
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Soraya Nishimura
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Rei Kashiwagi
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kaori Yasutake
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Miho Isoda
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Regenerative and Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd., Chuo-ku, Kobe 650-0047, Japan
| | - Morio Matsumoto
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Masaya Nakamura
- Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
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