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1
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Kuroda R, Niikura T, Matsumoto T, Fukui T, Oe K, Mifune Y, Minami H, Matsuoka H, Yakushijin K, Miyata Y, Kawamoto S, Kagimura T, Fujita Y, Kawamoto A. Phase III clinical trial of autologous CD34 + cell transplantation to accelerate fracture nonunion repair. BMC Med 2023; 21:386. [PMID: 37798633 PMCID: PMC10557317 DOI: 10.1186/s12916-023-03088-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2023] [Accepted: 09/21/2023] [Indexed: 10/07/2023] Open
Abstract
BACKGROUND We previously demonstrated that CD34 + cell transplantation in animals healed intractable fractures via osteogenesis and vasculogenesis; we also demonstrated the safety and efficacy of this cell therapy in an earlier phase I/II clinical trial conducted on seven patients with fracture nonunion. Herein, we present the results of a phase III clinical trial conducted to confirm the results of the previous phase studies using a larger cohort of patients. METHODS CD34 + cells were mobilized via administration of granulocyte colony-stimulating factor, harvested using leukapheresis, and isolated using magnetic cell sorting. Autologous CD34 + cells were transplanted in 15 patients with tibia nonunion and 10 patients with femur nonunion, who were followed up for 52 weeks post transplantation. The main outcome was a reduction in time to heal the tibia in nonunion patients compared with that in historical control patients. We calculated the required number of patients as 15 based on the results of the phase I/II study. An independent data monitoring committee performed the radiographic assessments. Adverse events and medical device failures were recorded. RESULTS All fractures healed during the study period. The time to radiological fracture healing was 2.8 times shorter in patients with CD34 + cell transplantation than in the historical control group (hazard ratio: 2.81 and 95% confidence interval 1.16-6.85); moreover, no safety concerns were observed. CONCLUSIONS Our findings strongly suggest that autologous CD34 + cell transplantation is a novel treatment option for fracture nonunion. TRIAL REGISTRATION UMIN-CTR, UMIN000022814. Registered on 22 June 2016.
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Affiliation(s)
- Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan
| | - Takahiro Niikura
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan.
| | - Tomoyuki Matsumoto
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan
| | - Tomoaki Fukui
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan
| | - Keisuke Oe
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan
| | - Yutaka Mifune
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan
| | - Hironobu Minami
- Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Hospital and Graduate School of Medicine, Chuo-Ku, Kobe, 650-0017, Japan
| | - Hiroshi Matsuoka
- Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Hospital and Graduate School of Medicine, Chuo-Ku, Kobe, 650-0017, Japan
| | - Kimikazu Yakushijin
- Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Hospital and Graduate School of Medicine, Chuo-Ku, Kobe, 650-0017, Japan
| | - Yoshiharu Miyata
- Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Hospital and Graduate School of Medicine, Chuo-Ku, Kobe, 650-0017, Japan
| | - Shinichiro Kawamoto
- Department of Transfusion Medicine and Cell Therapy, Kobe University Hospital, Chuo-Ku, Kobe, 650-0017, Japan
| | - Tatsuo Kagimura
- Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at Kobe, Chuo-Ku, Kobe, 650-0047, Japan
| | - Yasuyuki Fujita
- Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at Kobe, Chuo-Ku, Kobe, 650-0047, Japan
| | - Atsuhiko Kawamoto
- Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at Kobe, Chuo-Ku, Kobe, 650-0047, Japan
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Feehan J, Nurgali K, Apostolopoulos V, Duque G. Development and validation of a new method to isolate, expand, and differentiate circulating osteogenic precursor (COP) cells. Bone Rep 2021; 15:101109. [PMID: 34368409 PMCID: PMC8326352 DOI: 10.1016/j.bonr.2021.101109] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 07/21/2021] [Indexed: 11/17/2022] Open
Abstract
Circulating osteogenic precursor (COP) cells are a population of progenitor cells in the peripheral blood with the capacity to form bone in vitro and in vivo. They have characteristics of the mesenchymal stem and progenitor pool found in the bone marrow; however, more recently, a population of COP cells has been identified with markers of the hematopoietic lineage such as CD45 and CD34. While this population has been associated with several bone pathologies, a lack of cell culture models and inconsistent characterization has limited mechanistic research into their behavior and physiology. In this study, we describe a method for the isolation of CD45+/CD34+/alkaline phosphatase (ALP) + COP cells via fluorescence-activated cell sorting, as well as their expansion and differentiation in culture. Hematopoietic COP cells are a discreet population within the monocyte fraction of the peripheral blood mononuclear cells, which form proliferative, fibroblastoid colonies in culture. Their expression of hematopoietic markers decreases with time in culture, but they express markers of osteogenesis and deposit calcium with differentiation. It is hoped that this will provide a standard for their isolation, for consistency in future research efforts, to allow for the translation of COP cells into clinical settings.
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Affiliation(s)
- Jack Feehan
- Department of Medicine – Western Health, The University of Melbourne, Melbourne, Victoria, Australia
- Australian Institute of Musculoskeletal Science (AIMSS), The University of Melbourne, Western Health and Victoria University, Melbourne, Victoria, Australia
- Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia
| | - Kulmira Nurgali
- Department of Medicine – Western Health, The University of Melbourne, Melbourne, Victoria, Australia
- Australian Institute of Musculoskeletal Science (AIMSS), The University of Melbourne, Western Health and Victoria University, Melbourne, Victoria, Australia
- Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia
| | - Vasso Apostolopoulos
- Australian Institute of Musculoskeletal Science (AIMSS), The University of Melbourne, Western Health and Victoria University, Melbourne, Victoria, Australia
- Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia
| | - Gustavo Duque
- Department of Medicine – Western Health, The University of Melbourne, Melbourne, Victoria, Australia
- Australian Institute of Musculoskeletal Science (AIMSS), The University of Melbourne, Western Health and Victoria University, Melbourne, Victoria, Australia
- Institute for Health and Sport, Victoria University, Melbourne, Victoria, Australia
- Corresponding author at: Level 3, Western Centre for Health Research and Education, Sunshine Hospital, Furlong Road, St Albans, 3021 Melbourne, Australia.
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Oliveira CS, Carreira M, Correia CR, Mano JF. The Therapeutic Potential of Hematopoietic Stem Cells in Bone Regeneration. TISSUE ENGINEERING PART B-REVIEWS 2021; 28:379-392. [PMID: 33683146 DOI: 10.1089/ten.teb.2021.0019] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The repair process of bone fractures is a complex biological mechanism requiring the recruitment and in situ functionality of stem/stromal cells from the bone marrow (BM). BM mesenchymal stem/stromal cells have been widely explored in multiple bone tissue engineering applications, whereas the use of hematopoietic stem cells (HSCs) has been poorly investigated in this context. A reasonable explanation is the fact that the role of HSCs and their combined effect with other elements of the hematopoietic niches in the bone-healing process is still elusive. Therefore, in this review we intend to highlight the influence of HSCs in the bone repair process, mainly through the promotion of osteogenesis and angiogenesis at the bone injury site. For that, we briefly describe the main biological characteristics of HSCs, as well as their hematopoietic niches, while reviewing the biomimetic engineered BM niche models. Moreover, we also highlighted the role of HSCs in translational in vivo transplantation or implantation as promoters of bone tissue repair.
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Affiliation(s)
- Cláudia S Oliveira
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
| | - Mariana Carreira
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
| | - Clara R Correia
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
| | - João F Mano
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
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Kamenaga T, Kuroda Y, Nagai K, Tsubosaka M, Takashima Y, Kikuchi K, Fujita M, Ikuta K, Anjiki K, Maeda T, Nakano N, Takayama K, Hashimoto S, Hayashi S, Matsushita T, Niikura T, Kuroda R, Matsumoto T. Cryopreserved human adipose-derived stromal vascular fraction maintains fracture healing potential via angiogenesis and osteogenesis in an immunodeficient rat model. Stem Cell Res Ther 2021; 12:110. [PMID: 33541427 PMCID: PMC7863470 DOI: 10.1186/s13287-021-02182-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Accepted: 01/24/2021] [Indexed: 12/20/2022] Open
Abstract
Background Novel therapeutic strategies for the healing of nonunion, which has serious effects on the quality of life of patients, are needed. We evaluated the therapeutic effect of local transplantation of human stromal vascular fraction (SVF) cells on fracture healing in a rat non-healing fracture model and compared the effects between freshly isolated (F) and cryopreserved (C)-SVFs. Methods Non-healing fracture model was induced in the femur of female immunodeficient rats (F344/N Jcl rnu/rnu) with cauterizing periosteum. Immediately after the creation of non-healing fracture, rats received local transplantation of F and C-SVFs suspended in phosphate-buffered saline (PBS) or the same volume of PBS without cells using the same scaffold as a control group. During 8 weeks post-surgery, radiologic, histological, immunohistochemical, and biomechanical analyses were performed to evaluate fracture healing. The comparison of radiological results was performed with a chi-square test, and the multiple comparisons of immunohistochemical, histological, and biomechanical results among groups were made using a one-way analysis of variance. A probability value of 0.05 was considered to denote statistical significance. Results At week 8, in 60% of animals receiving F-SVF cells and in 50% of animals receiving C-SVF cells, the fracture radiologically healed with bone union whereas nonunion was observed in the control group. The healing potential was also confirmed by histological and biomechanical assessments. One of the mechanisms underlying healing involving intrinsic angiogenesis/osteogenesis was enhanced in F- and C-SVF groups compared with that in the control group. Human cell-derived vasculogenesis/osteogenesis, which was also confirmed in an in vitro differentiation assay, was also enhanced in the F- and C-SVF groups compared with that in the control groups and could be another mechanism for healing. Conclusions SVF cells can enhance bone healing and cryopreserved cells have almost equal potential as fresh cells. SVF cells can be used for improving nonunion bone fracture healing as an alternative to other mesenchymal stem cells and the effect of SVF cells can be maintained under cryopreservation.
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Affiliation(s)
- Tomoyuki Kamenaga
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Yuichi Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Kanto Nagai
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Masanori Tsubosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Yoshinori Takashima
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Kenichi Kikuchi
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Masahiro Fujita
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Kemmei Ikuta
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Kensuke Anjiki
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Toshihisa Maeda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Naoki Nakano
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Koji Takayama
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Shingo Hashimoto
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Shinya Hayashi
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Takehiko Matsushita
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Takahiro Niikura
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan
| | - Tomoyuki Matsumoto
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-chou, 650-0017, Kobe, Japan.
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Feehan J, Kassem M, Pignolo RJ, Duque G. Bone From Blood: Characteristics and Clinical Implications of Circulating Osteogenic Progenitor (COP) Cells. J Bone Miner Res 2021; 36:12-23. [PMID: 33118647 DOI: 10.1002/jbmr.4204] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Revised: 10/22/2020] [Accepted: 10/23/2020] [Indexed: 02/06/2023]
Abstract
Circulating osteogenic progenitor (COP) cells are a population of cells in the peripheral blood with the capacity for bone formation, as well as broader differentiation into mesoderm-like cells in vitro. Although some of their biological characteristics are documented in vitro, their role in diseases of the musculoskeletal system remains yet to be fully evaluated. In this review, we provide an overview of the role of COP cells in a number of physiological and pathological conditions, as well as identify areas for future research. In addition, we suggest possible areas for clinical utilization in the management of musculoskeletal diseases. © 2020 American Society for Bone and Mineral Research (ASBMR).
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Affiliation(s)
- Jack Feehan
- Australian Institute for Musculoskeletal Science (AIMSS), University of Melbourne and Western Health, St Albans, VIC, Australia.,Department of Medicine, University of Melbourne-Western Health, Melbourne, VIC, Australia
| | - Moustapha Kassem
- Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University Hospital & University of Southern Denmark, Odense, Denmark.,Department of Cellular and Molecular Medicine, The Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), University of Copenhagen, Copenhagen, Denmark
| | - Robert J Pignolo
- Department of Medicine, Mayo Clinic School of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Gustavo Duque
- Australian Institute for Musculoskeletal Science (AIMSS), University of Melbourne and Western Health, St Albans, VIC, Australia.,Department of Medicine, University of Melbourne-Western Health, Melbourne, VIC, Australia
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6
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Kargozar S, Mozafari M, Hamzehlou S, Brouki Milan P, Kim HW, Baino F. Bone Tissue Engineering Using Human Cells: A Comprehensive Review on Recent Trends, Current Prospects, and Recommendations. APPLIED SCIENCES 2019; 9:174. [DOI: 10.3390/app9010174] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The use of proper cells for bone tissue engineering remains a major challenge worldwide. Cells play a pivotal role in the repair and regeneration of the bone tissue in vitro and in vivo. Currently, a large number of differentiated (somatic) and undifferentiated (stem) cells have been used for bone reconstruction alone or in combination with different biomaterials and constructs (e.g., scaffolds). Although the results of the cell transplantation without any supporting or adjuvant material have been very effective with regard to bone healing. Recent advances in bone scaffolding are now becoming new players affecting the osteogenic potential of cells. In the present study, we have critically reviewed all the currently used cell sources for bone reconstruction and discussed the new horizons that are opening up in the context of cell-based bone tissue engineering strategies.
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Affiliation(s)
- Saeid Kargozar
- Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad 917794-8564, Iran
| | - Masoud Mozafari
- Bioengineering Research Group, Nanotechnology and Advanced Materials Department, Materials and Energy Research Center (MERC), Tehran 14155-4777, Iran
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS), Tehran 144961-4535, Iran
| | - Sepideh Hamzehlou
- Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran 14155-6447, Iran
- Medical Genetics Network (MeGeNe), Universal Scientific Education and Research Network (USERN), Tehran, Iran
| | - Peiman Brouki Milan
- Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS), Tehran 144961-4535, Iran
| | - Hae-Won Kim
- Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 31116, Korea
- Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 31116, Korea
- Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine Research Center, Dankook University, Cheonan 31116, Korea
| | - Francesco Baino
- Institute of Materials Physics and Engineering, Applied Science and Technology Department, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy
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Hirashima S, Ohta K, Hagihara M, Shimizu M, Kanazawa T, Nakamura KI. Effects of an in Vitro Reconstructed Three-dimensional Hematopoietic Microenvironment on Bone Regeneration in a Rat Calvarial Defect Model. J HARD TISSUE BIOL 2018. [DOI: 10.2485/jhtb.27.185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Affiliation(s)
- Shingo Hirashima
- Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine
- Dental and Oral Medical Center, Kurume University School of Medicine
| | - Keisuke Ohta
- Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine
- Advanced Imaging Research Center, Kurume University School of Medicine
| | - Masahiko Hagihara
- Ube Industries, Ltd. Corporate Research and Development, Hagihara Research Group
| | - Motohisa Shimizu
- Ube Industries, Ltd. Corporate Research and Development, Hagihara Research Group
| | - Tomonoshin Kanazawa
- Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine
| | - Kei-ichiro Nakamura
- Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine
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Zhang Y, Husch JFA, van den Beucken JJJP. Intraoperative Construct Preparation: A Practical Route for Cell-Based Bone Regeneration. TISSUE ENGINEERING PART B-REVIEWS 2018; 24:403-417. [PMID: 29631489 DOI: 10.1089/ten.teb.2018.0010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Stem cell-based bone tissue engineering based on the combination of a scaffold and expanded autologous mesenchymal stem cells (MSCs) represents the current state-of-the-art treatment for bone defects and fractures. However, the procedure of such construct preparation requires extensive ex vivo manipulation of patient's cells to achieve enough stem cells. Therefore, it is impractical and not cost-effective compared to other therapeutic interventions. For these reasons, a more practical strategy circumventing any ex vivo manipulation and an additional surgery for the patient would be advantageous. Intraoperative concept-based bone tissue engineering, where constructs are prepared with easily accessible autologous cells within the same surgical procedure, allows for such a simplification. In this study, we discuss the concept of intraoperative construct preparation for bone tissue engineering and summarize the available cellular options for intraoperative preparation. Furthermore, we propose methods to prepare intraoperative constructs, and review data of currently available preclinical and clinical studies using intraoperatively prepared constructs for bone regenerative applications. We identify several obstacles hampering the application of this emerging approach and highlight perspectives of technological innovations to advance the future developments of intraoperative construct preparation.
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Affiliation(s)
- Yang Zhang
- Department of Biomaterials, Radboudumc, Nijmegen, The Netherlands
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9
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Herrmann M, Zeiter S, Eberli U, Hildebrand M, Camenisch K, Menzel U, Alini M, Verrier S, Stadelmann VA. Five Days Granulocyte Colony-Stimulating Factor Treatment Increases Bone Formation and Reduces Gap Size of a Rat Segmental Bone Defect: A Pilot Study. Front Bioeng Biotechnol 2018; 6:5. [PMID: 29484293 PMCID: PMC5816045 DOI: 10.3389/fbioe.2018.00005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2017] [Accepted: 01/15/2018] [Indexed: 12/25/2022] Open
Abstract
Bone is an organ with high natural regenerative capacity and most fractures heal spontaneously when appropriate fracture fixation is provided. However, additional treatment is required for patients with large segmental defects exceeding the endogenous healing potential and for patients suffering from fracture non-unions. These cases are often associated with insufficient vascularization. Transplantation of CD34+ endothelial progenitor cells (EPCs) has been successfully applied to promote neovascularization of bone defects, however including extensive ex vivo manipulation of cells. Here, we hypothesized, that treatment with granulocyte colony-stimulating factor (G-CSF) may improve bone healing by mobilization of CD34+ progenitor cells into the circulation, which in turn may facilitate vascularization at the defect site. In this pilot study, we aimed to characterize the different cell populations mobilized by G-CSF and investigate the influence of cell mobilization on the healing of a critical size femoral defect in rats. Cell mobilization was investigated by flow cytometry at different time points after five consecutive daily G-CSF injections. In a pilot study, bone healing of a 4.5-mm critical femoral defect in F344 rats was compared between a saline-treated control group and a G-CSF treatment group. In vivo microcomputed tomography and histology were applied to compare bone formation in both treatment groups. Our data revealed that leukocyte counts show a peak increase at the first day after the last G-CSF injection. In addition, we found that CD34+ progenitor cells, including EPCs, were significantly enriched at day 1, and further increased at day 5 and day 11. Upregulation of monocytes, granulocytes and macrophages peaked at day 1. G-CSF treatment significantly increased bone volume and bone density in the defect, which was confirmed by histology. Our data show that different cell populations are mobilized by G-CSF treatment in cell specific patterns. Although in this pilot study no bridging of the critical defect was observed, significantly improved bone formation by G-CSF treatment was clearly shown.
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Affiliation(s)
| | | | | | | | | | | | - Mauro Alini
- AO Research Institute Davos, Davos, Switzerland
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10
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Mifuji K, Ishikawa M, Kamei N, Tanaka R, Arita K, Mizuno H, Asahara T, Adachi N, Ochi M. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017; 6:489-498. [PMID: 28835445 PMCID: PMC5579315 DOI: 10.1302/2046-3758.68.bjr-2016-0338.r1] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2017] [Accepted: 05/08/2017] [Indexed: 12/22/2022] Open
Abstract
Objectives The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. Results The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. Conclusion We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion. Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1.
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Affiliation(s)
- K Mifuji
- Hiroshima University, Hiroshima, Japan
| | | | - N Kamei
- Hiroshima University, Hiroshima, Japan
| | - R Tanaka
- Juntendo University School of Medicine, Tokyo, Japan
| | - K Arita
- Juntendo University School of Medicine, Tokyo, Japan
| | - H Mizuno
- Juntendo University School of Medicine, Tokyo, Japan
| | - T Asahara
- Tokai University School of Medicine, Isehara, Kanagawa, Japan
| | - N Adachi
- Hiroshima University, Hiroshima, Japan
| | - M Ochi
- Hiroshima University, Hiroshima, Japan
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11
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Hertweck J, Ritz U, Götz H, Schottel PC, Rommens PM, Hofmann A. CD34 + cells seeded in collagen scaffolds promote bone formation in a mouse calvarial defect model. J Biomed Mater Res B Appl Biomater 2017; 106:1505-1516. [PMID: 28730696 DOI: 10.1002/jbm.b.33956] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Revised: 05/29/2017] [Accepted: 07/04/2017] [Indexed: 11/10/2022]
Abstract
Bone tissue engineering (BTE) holds promise for managing the clinical problem of large bone defects. However, clinical adoption of BTE is limited due to limited vascularization of constructs, which could be circumvented by pre-cultivation of osteogenic and endothelial derived cells in natural-based polymer scaffolds. However, until now not many studies compared the effect of mono- and cocultures pre-seeded in collagen before implantation. We utilized a mouse calvarial defect model and compared five groups of collagen scaffolds: a negative control of a collagen scaffold alone, a positive control treated with BMP-7, monocultures of either human osteoblasts (hOBs) or CD34+ cells, and a coculture of hOB and CD34+ cells. Each pre-seeded collagen scaffold was implanted in mice. After 6 weeks mice were sacrificed and their skulls prepared for volumetric and histologic analysis. We found that a monoculture of CD34+ cells and a coculture of hOB and CD34+ cells pre-cultured in the collagen scaffold increased bone regeneration to a similar extend. In these groups, greater amounts of new bone were found compared with hOB monocultures. Interestingly, monoculture of CD34+ cells demonstrated better fracture healing than monoculture of hOBs, emphasizing the possible role of angiogenesis. Our results are promising regarding a cellular based collagen BTE construct, but more work is needed to understand the complex interaction between the osteogenic and endothelial cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1505-1516, 2018.
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Affiliation(s)
- Jens Hertweck
- Department of Orthopaedics and Traumatology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Ulrike Ritz
- Department of Orthopaedics and Traumatology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Hermann Götz
- Platform for Biomaterial Research, Biomatics, University Medical Centre, Johannes Gutenberg University, Mainz, Germany
| | - Patrick C Schottel
- Department of Orthopedics and Rehabilitation, University of Vermont Medical Center, Burlington, Vermont
| | - Pol Maria Rommens
- Department of Orthopaedics and Traumatology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Alexander Hofmann
- Department of Orthopaedics and Traumatology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany
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12
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The Use of Endothelial Progenitor Cells for the Regeneration of Musculoskeletal and Neural Tissues. Stem Cells Int 2017; 2017:1960804. [PMID: 28458693 PMCID: PMC5387841 DOI: 10.1155/2017/1960804] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2016] [Accepted: 03/12/2017] [Indexed: 12/18/2022] Open
Abstract
Endothelial progenitor cells (EPCs) derived from bone marrow and blood can differentiate into endothelial cells and promote neovascularization. In addition, EPCs are a promising cell source for the repair of various types of vascularized tissues and have been used in animal experiments and clinical trials for tissue repair. In this review, we focused on the kinetics of endogenous EPCs during tissue repair and the application of EPCs or stem cell populations containing EPCs for tissue regeneration in musculoskeletal and neural tissues including the bone, skeletal muscle, ligaments, spinal cord, and peripheral nerves. EPCs can be mobilized from bone marrow and recruited to injured tissue to contribute to neovascularization and tissue repair. In addition, EPCs or stem cell populations containing EPCs promote neovascularization and tissue repair through their differentiation to endothelial cells or tissue-specific cells, the upregulation of growth factors, and the induction and activation of endogenous stem cells. Human peripheral blood CD34(+) cells containing EPCs have been used in clinical trials of bone repair. Thus, EPCs are a promising cell source for the treatment of musculoskeletal and neural tissue injury.
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Harrell DB, Caradonna E, Mazzucco L, Gudenus R, Amann B, Prochazka V, Giannoudis PV, Hendrich C, Jäger M, Krauspe R, Hernigou P. Non-Hematopoietic Essential Functions of Bone Marrow Cells: A Review of Scientific and Clinical Literature and Rationale for Treating Bone Defects. Orthop Rev (Pavia) 2015; 7:5691. [PMID: 26793290 PMCID: PMC4703908 DOI: 10.4081/or.2015.5691] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2014] [Accepted: 10/20/2015] [Indexed: 01/13/2023] Open
Abstract
Hematopoiesis as the only essential function of bone marrow cells has been challenged for several decades through basic science (in vitro and in vivo) and clinical data. Such work has shed light on two other essential functions of bone marrow cells: osteopoiesis and angio-genesis/vasculogenesis. Clinical utility of autologous concentrated bone marrow aspirate (CBMA) has demonstrated both safety and efficacy in treating bone defects. Moreover, CBMA has been shown to be comparable to the gold standard of iliac crest bone graft (ICBG), or autograft, with regard to being osteogenic and osteoinductive. ICBG is not considered an advanced therapy medicinal product (ATMP), but CBMA may become regulated as an ATMP. The European Medicines Agency Committee for Advanced Therapies (EMA:CAT) has issued a reflection paper (20 June 2014) in which reversal of the 2013 ruling that CBMA is a non-ATMP has been proposed. We review bone marrow cell involvement in osteopoiesis and angiogenesis/vasculogenesis to examine EMA:CAT 2013 decision to use CBMA for treatment of osteonecrosis (e.g, of the femoral head) should be considered a non-ATMP. This paper is intended to provide discussion on the 20 June 2014 reflection paper by reviewing two non-hematopoietic essential functions of bone marrow cells. Additionally, we provide clinical and scientific rationale for treating osteonecrosis with CBMA.
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Affiliation(s)
| | - Eugenio Caradonna
- Department of Cardiovascular Disease, Fondazione de Ricerca e Cura Giovanni e Paolo II, Campbasso, Italy
| | - Laura Mazzucco
- Blood Component and Regenerative Medicine Laboratory, Alessandria Hospital, Italy
| | | | | | - Vaclav Prochazka
- Interventional Neuroradiology and Angiology, University of Ostrava, Czech Republic
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14
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Zhang S, Matsumoto T, Uefuji A, Matsushita T, Takayama K, Araki D, Nakano N, Nagai K, Matsuzaki T, Kuroda R, Kurosaka M. Anterior cruciate ligament remnant tissue harvested within 3-months after injury predicts higher healing potential. BMC Musculoskelet Disord 2015; 16:390. [PMID: 26687109 PMCID: PMC4684911 DOI: 10.1186/s12891-015-0855-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2015] [Accepted: 12/16/2015] [Indexed: 11/10/2022] Open
Abstract
Background No study has examined the possible factors associated with different characteristics of stem-like cells derived from anterior cruciate ligament (ACL) remnants. And the purpose of the study is to elucidate whether demographic factors are associated with healing potential of stem-like cells derived from the ACL remnants tissue. Methods Thirty-six ACL remnants were harvested from patients who received primary arthroscopic ACL reconstruction. Interval from injury to surgery, age, sex, and combined meniscal or chondral injuries were analyzed. Cells were isolated from remnant tissues and their healing potential was evaluated by: 1) characterization of surface markers (CD34, CD44, CD45, CD146, CD29, and Stro-1), 2) cell expansion, 3) osteogenic differentiation, and 4) endothelial differentiation. Finally, using multivariable logistic regression to evaluate the relation between demographic factors and healing potential parameters. Adjusted odds ratios (OR) were calculated, and the significant difference was set at p < 0.05. Results ACL remnant tissue harvested less than 90 days after injury predicted higher fractions of stem-like cells [CD34+ (OR = 6.043, p = 0.025), CD44 + (OR = 8.440, p = 0.011), CD45+ (OR = 16.144, p = 0.015), and CD146+ (OR = 9.246, p = 0.015)] and higher expansion potential (passage 3: OR = 9.755, p = 0.034; passage 10: OR = 33.245, p = 0.003). Regarding osteogenic differentiation, higher gene expression of Osteocalcin (OR = 22.579, p = 0.009), Alkaline phosphatase (OR = 6.527, p = 0.022), and Runt-related transcription factor 2 (OR = 5.247, p = 0.047) can also be predicted. Younger age predicted higher CD34+ levels (20 ≤ age <30 years, OR = 2.020, p = 0.027) and higher expansion potential at passage 10 (10 ≤ age <20 years, OR = 25.141, p = 0.026). There was no significant relation found between meniscal or chondral injuries and ACL healing potential. Conclusion Our results indicated that the ACL remnant tissue harvested within 3-months after injury yields higher healing potential, suggesting early surgical intervention may achieve better clinical results.
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Affiliation(s)
- Shurong Zhang
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Tomoyuki Matsumoto
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Atsuo Uefuji
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Takehiko Matsushita
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Koji Takayama
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Daisuke Araki
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Naoki Nakano
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Kanto Nagai
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Tokio Matsuzaki
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
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Abstract
Stem cells offer great promise to help understand the normal mechanisms of tissue renewal, regeneration, and repair, and also for development of cell-based therapies to treat patients after tissue injury. Most adult tissues contain stem cells and progenitor cells that contribute to homeostasis, remodeling, and repair. Multiple stem and progenitor cell populations in bone are found in the marrow, the endosteum, and the periosteum. They contribute to the fracture healing process after injury and are an important component in tissue engineering approaches for bone repair. This review focuses on current concepts in stem cell biology related to fracture healing and bone tissue regeneration, as well as current strategies and limitations for clinical cell-based therapies.
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16
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Gamie Z, MacFarlane RJ, Tomkinson A, Moniakis A, Tran GT, Gamie Y, Mantalaris A, Tsiridis E. Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data. Expert Opin Biol Ther 2015; 14:1611-39. [PMID: 25303322 DOI: 10.1517/14712598.2014.945414] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
INTRODUCTION Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources. AREAS COVERED In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases. EXPERT OPINION Enhanced results have been found when combining bone marrow-derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue-derived stem cells and umbilical cord tissue-derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.
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Affiliation(s)
- Zakareya Gamie
- Aristotle University Medical School, 'PapaGeorgiou' Hospital, Academic Orthopaedic Unit , Thessaloniki , Greece
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Fukui T, Mifune Y, Matsumoto T, Shoji T, Kawakami Y, Kawamoto A, Ii M, Akimaru H, Kuroda T, Horii M, Yokoyama A, Alev C, Kuroda R, Kurosaka M, Asahara T. Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion following Bone Fracture. Cell Transplant 2015; 24:1379-93. [DOI: 10.3727/096368914x681586] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
We recently demonstrated that the local transplantation of human peripheral blood (PB) CD34+ cells, an endothelial/hematopoietic progenitor cell-rich population, contributes to fracture repair via vasculogenesis/angiogenesis and osteogenesis. Human PB mononuclear cells (MNCs) are also considered a potential cell fraction for neovascularization. We have previously shown the feasibility of human PB MNCs to enhance fracture healing. However, there is no report directly comparing the efficacy for fracture repair between CD34+ cells and MNCs. In addition, an unhealing fracture model, which does not accurately resemble a clinical setting, was used in our previous studies. To overcome these issues, we compared the capacity of human granulocyte colony-stimulating factor-mobilized PB (GM-PB) CD34+ cells and human GM-PB MNCs in a nonunion model, which more closely resembles a clinical setting. First, the effect of local transplantation of 1 × 105 GM-PB CD34+ cells (CD34+ group), 1 × 107 GM-PB MNCs (containing approximately 1 × 105 GM-PB CD34+ cells) (MNC group), and phosphate-buffered saline (PBS) (PBS group) on nonunion healing was compared. Similar augmentation of blood flow recovery at perinonunion sites was observed in the CD34+ and MNC groups. Meanwhile, a superior effect on nonunion repair was revealed by radiological, histological, and functional assessment in the CD34+ group compared with the other groups. Moreover, through in vivo and in vitro experiments, excessive inflammation induced by GM-PB MNCs was confirmed and believed to be one of the mechanisms underlying this potency difference. These results strongly suggest that local transplantation of GM-PB CD34+ cells is a practical and effective strategy for treatment of nonunion after fracture.
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Affiliation(s)
- Tomoaki Fukui
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Yutaka Mifune
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoyuki Matsumoto
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Taro Shoji
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Yohei Kawakami
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Atsuhiko Kawamoto
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Masaaki Ii
- Group of Translational Stem Cell Research, Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan
| | - Hiroshi Akimaru
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Tomoya Kuroda
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Miki Horii
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Ayumi Yokoyama
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Cantas Alev
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Takayuki Asahara
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
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Herrmann M, Verrier S, Alini M. Strategies to Stimulate Mobilization and Homing of Endogenous Stem and Progenitor Cells for Bone Tissue Repair. Front Bioeng Biotechnol 2015; 3:79. [PMID: 26082926 PMCID: PMC4451737 DOI: 10.3389/fbioe.2015.00079] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2015] [Accepted: 05/16/2015] [Indexed: 12/17/2022] Open
Abstract
The gold standard for the treatment of critical-size bone defects is autologous or allogenic bone graft. This has several limitations including donor site morbidity and the restricted supply of graft material. Cell-based tissue engineering strategies represent an alternative approach. Mesenchymal stem cells (MSCs) have been considered as a source of osteoprogenitor cells. More recently, focus has been placed on the use of endothelial progenitor cells (EPCs), since vascularization is a critical step in bone healing. Although many of these approaches have demonstrated effectiveness for bone regeneration, cell-based therapies require time consuming and cost-expensive in vitro cell expansion procedures. Accordingly, research is becoming increasingly focused on the homing and stimulation of native cells. The stromal cell-derived factor-1 (SDF-1) - CXCR4 axis has been shown to be critical for the recruitment of MSCs and EPCs. Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis and has been targeted in many studies. Here, we present an overview of the different approaches for delivering homing factors to the defect site by absorption or incorporation to biomaterials, gene therapy, or via genetically manipulated cells. We further review strategies focusing on the stimulation of endogenous cells to support bone repair. Finally, we discuss the major challenges in the treatment of critical-size bone defects and fracture non-unions.
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Affiliation(s)
| | | | - Mauro Alini
- AO Research Institute Davos , Davos , Switzerland
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19
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Zigdon-Giladi H, Rudich U, Michaeli Geller G, Evron A. Recent advances in bone regeneration using adult stem cells. World J Stem Cells 2015; 7:630-640. [PMID: 25914769 PMCID: PMC4404397 DOI: 10.4252/wjsc.v7.i3.630] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 12/30/2014] [Accepted: 01/20/2015] [Indexed: 02/06/2023] Open
Abstract
Bone is a highly vascularized tissue reliant on the close spatial and temporal association between blood vessels and bone cells. Therefore, cells that participate in vasculogenesis and osteogenesis play a pivotal role in bone formation during prenatal and postnatal periods. Nevertheless, spontaneous healing of bone fracture is occasionally impaired due to insufficient blood and cellular supply to the site of injury. In these cases, bone regeneration process is interrupted, which might result in delayed union or even nonunion of the fracture. Nonunion fracture is difficult to treat and have a high financial impact. In the last decade, numerous technological advancements in bone tissue engineering and cell-therapy opened new horizon in the field of bone regeneration. This review starts with presentation of the biological processes involved in bone development, bone remodeling, fracture healing process and the microenvironment at bone healing sites. Then, we discuss the rationale for using adult stem cells and listed the characteristics of the available cells for bone regeneration. The mechanism of action and epigenetic regulations for osteogenic differentiation are also described. Finally, we review the literature for translational and clinical trials that investigated the use of adult stem cells (mesenchymal stem cells, endothelial progenitor cells and CD34+ blood progenitors) for bone regeneration.
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20
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Paul AJ, Momier D, Boukhechba F, Michiels JF, Lagadec P, Rochet N. Effect of G-CSF on the osteoinductive property of a BCP/blood clot composite. J Biomed Mater Res A 2015; 103:2830-8. [DOI: 10.1002/jbm.a.35424] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2014] [Revised: 01/19/2015] [Accepted: 02/04/2015] [Indexed: 11/11/2022]
Affiliation(s)
- Adrien J. Paul
- Université Nice Sophia Antipolis, iBV, UMR7277; Nice 06100 France
- CNRS, iBV, UMR7277; Nice 06100 France
- Inserm, iBV, U1091; Nice 06100 France
- Université Nice Sophia Antipolis, UFR odontologie; Nice 06000 France
- Centre Hospitalier Universitaire, Pôle d'odontologie; Nice 06000 France
| | - David Momier
- Université Nice Sophia Antipolis, iBV, UMR7277; Nice 06100 France
- CNRS, iBV, UMR7277; Nice 06100 France
- Inserm, iBV, U1091; Nice 06100 France
| | - Florian Boukhechba
- Université Nice Sophia Antipolis, iBV, UMR7277; Nice 06100 France
- CNRS, iBV, UMR7277; Nice 06100 France
- Inserm, iBV, U1091; Nice 06100 France
- Graftys, 13854 Aix En Provence; France
| | | | - Patricia Lagadec
- Université Nice Sophia Antipolis, iBV, UMR7277; Nice 06100 France
- CNRS, iBV, UMR7277; Nice 06100 France
- Inserm, iBV, U1091; Nice 06100 France
| | - Nathalie Rochet
- Université Nice Sophia Antipolis, iBV, UMR7277; Nice 06100 France
- CNRS, iBV, UMR7277; Nice 06100 France
- Inserm, iBV, U1091; Nice 06100 France
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Kawakami Y, Ii M, Matsumoto T, Kuroda R, Kuroda T, Kwon SM, Kawamoto A, Akimaru H, Mifune Y, Shoji T, Fukui T, Kurosaka M, Asahara T. SDF-1/CXCR4 axis in Tie2-lineage cells including endothelial progenitor cells contributes to bone fracture healing. J Bone Miner Res 2015; 30:95-105. [PMID: 25130304 DOI: 10.1002/jbmr.2318] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2014] [Revised: 06/30/2014] [Accepted: 07/25/2014] [Indexed: 12/13/2022]
Abstract
CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal-derived-factor 1 (SDF-1). SDF-1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF-1/CXCR4 pathway in Tie2-lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2-Cre(ER) CXCR4 knockout mice (CXCR4(-/-) ) and wild-type mice (WT). We report here that in vitro, EPCs derived from of CXCR4(-/-) mouse bone marrow demonstrated severe reduction of migration activity and EPC colony-forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4(-/-) group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4(-/-) group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4(-/-) group. Especially, CXCR4(-/-) group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real-time RT-PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE-cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4(-/-) group. In the gain-of-function study, the fracture in the SDF-1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF-1 injected CXCR4(-/-) group. We demonstrated that an EPC SDF-1/CXCR4 axis plays an important role in bone fracture healing using Tie2-Cre(ER) CXCR4 conditional knockout mice.
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Affiliation(s)
- Yohei Kawakami
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Japan; Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
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22
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Tam WL, O DF, Hiramatsu K, Tsumaki N, Luyten FP, Roberts SJ. Sox9 reprogrammed dermal fibroblasts undergo hypertrophic differentiation in vitro and trigger endochondral ossification in vivo. Cell Reprogram 2014; 16:29-39. [PMID: 24459991 DOI: 10.1089/cell.2013.0060] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies.
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Affiliation(s)
- Wai Long Tam
- 1 Laboratory for Developmental and Stem Cell Biology (DSB) , Skeletal Biology and Engineering Research Center (SBE), KU Leuven, 3000, Leuven, Belgium
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Bone marrow derived stem cells in joint and bone diseases: a concise review. INTERNATIONAL ORTHOPAEDICS 2014; 38:1787-801. [PMID: 25005462 DOI: 10.1007/s00264-014-2445-4] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2014] [Accepted: 06/21/2014] [Indexed: 12/11/2022]
Abstract
Stem cells have huge applications in the field of tissue engineering and regenerative medicine. Their use is currently not restricted to the life-threatening diseases but also extended to disorders involving the structural tissues, which may not jeopardize the patients' life, but certainly influence their quality of life. In fact, a particularly popular line of research is represented by the regeneration of bone and cartilage tissues to treat various orthopaedic disorders. Most of these pioneering research lines that aim to create new treatments for diseases that currently have limited therapies are still in the bench of the researchers. However, in recent years, several clinical trials have been started with satisfactory and encouraging results. This article aims to review the concept of stem cells and their characterization in terms of site of residence, differentiation potential and therapeutic prospective. In fact, while only the bone marrow was initially considered as a "reservoir" of this cell population, later, adipose tissue and muscle tissue have provided a considerable amount of cells available for multiple differentiation. In reality, recently, the so-called "stem cell niche" was identified as the perivascular space, recognizing these cells as almost ubiquitous. In the field of bone and joint diseases, their potential to differentiate into multiple cell lines makes their application ideally immediate through three main modalities: (1) cells selected by withdrawal from bone marrow, subsequent culture in the laboratory, and ultimately transplant at the site of injury; (2) bone marrow aspirate, concentrated and directly implanted into the injury site; (3) systemic mobilization of stem cells and other bone marrow precursors by the use of growth factors. The use of this cell population in joint and bone disease will be addressed and discussed, analysing both the clinical outcomes but also the basic research background, which has justified their use for the treatment of bone, cartilage and meniscus tissues.
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Uefuji A, Matsumoto T, Matsushita T, Ueha T, Zhang S, Kurosaka M, Kuroda R. Age-Related Differences in Anterior Cruciate Ligament Remnant Vascular-Derived Cells. Am J Sports Med 2014; 42:1478-86. [PMID: 24727934 DOI: 10.1177/0363546514529092] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
BACKGROUND The anterior cruciate ligament (ACL) does not heal spontaneously after injury, and patients of different ages respond differently to treatment. CD34+ stem/progenitor cells derived from the ACL remnant and associated tissues contribute to tendon-bone healing, but the relationship between age and the ACL's healing potential has not been clarified. HYPOTHESIS The ACL remnant and associated tissues from adolescent patients have more CD34+ cells, and this population of cells from younger patients exhibits a higher potential for proliferation and differentiation in vitro. STUDY DESIGN Descriptive laboratory study. METHODS Ruptured ACL remnants and associated tissues were harvested from 28 patients (mean age, 24.6 ± 1.6 years) who had undergone primary arthroscopic ACL reconstruction. Patients were divided into 3 patient groups by age: 10-19 years (teens group; n = 10), 20-29 years (20s group; n = 10), and ≥30 years (30s group; n = 8). The ACL remnant cells were characterized using fluorescence-activated cell sorting (FACS). Expansion potential was evaluated using population doubling (PD), and multilineage differentiation potential was assessed and compared. RESULTS The FACS analysis showed numerous CD34+ cells in the teens group compared with the 30s group (mean, 25.4% ± 7.9% vs 16.9% ± 3.9%, respectively; P = .044). The PD results indicated that the teens group had a significantly higher expansion potential than the 30s group at passage 3 (mean, 3.3 ± 0.2 vs 2.8 ± 0.2, respectively; P = .039). Young ACL remnant cells had a higher potential for osteogenic differentiation according to alkaline phosphatase activity (teens group, 169.5 ± 37.9 × 10 ng/mL vs 30s group, 64.9 ± 14.6 × 10 ng/mL; P = .029) and osteocalcin gene expression (teens group, 1.0 ± 0.25 vs 30s group, 0.39 ± 0.01; P = .01). In addition, the teens group displayed a higher differentiation potential to angiogenic lineages (acetylated low-density lipoprotein/Ulex europaeus lectin-stained cell counts) than other groups (teens group, 15.9 ± 1.9 vs 20s group, 8.9 ± 1.3 [P = .04]; teens group, 15.9 ± 1.9 vs 30s group, 7.2 ± 1.5 [P = .008]) and also tube length (teens group, 6939 ± 470 μm vs 30s group, 4119 ± 507 μm; P = .009). CONCLUSION The ACL remnants of adolescent patients had more CD34+ cells, and those cells had a higher potential for proliferation and multilineage differentiation in vitro. CLINICAL RELEVANCE During remnant-preserving or remnant-transplanted ACL reconstruction, surgeons should consider the patient's age when predicting the healing potential.
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Affiliation(s)
- Atsuo Uefuji
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Tomoyuki Matsumoto
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Takehiko Matsushita
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
| | | | - Shurong Zhang
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
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Controlled release of granulocyte colony-stimulating factor enhances osteoconductive and biodegradable properties of Beta-tricalcium phosphate in a rat calvarial defect model. Int J Biomater 2014; 2014:134521. [PMID: 24829581 PMCID: PMC4009298 DOI: 10.1155/2014/134521] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2013] [Revised: 11/27/2013] [Accepted: 11/27/2013] [Indexed: 12/12/2022] Open
Abstract
Autologous bone grafts remain the gold standard for the treatment of congenital craniofacial disorders; however, there are potential problems including donor site morbidity and limitations to the amount of bone that can be harvested. Recent studies suggest that granulocyte colony-stimulating factor (G-CSF) promotes fracture healing or osteogenesis. The purpose of the present study was to investigate whether topically applied G-CSF can stimulate the osteoconductive properties of beta-tricalcium phosphate (β-TCP) in a rat calvarial defect model. A total of 27 calvarial defects 5 mm in diameter were randomly divided into nine groups, which were treated with various combinations of a β-TCP disc and G-CSF in solution form or controlled release system using gelatin hydrogel. Histologic and histomorphometric analyses were performed at eight weeks postoperatively. The controlled release of low-dose (1 μg and 5 μg) G-CSF significantly enhanced new bone formation when combined with a β-TCP disc. Moreover, administration of 5 μg G-CSF using a controlled release system significantly promoted the biodegradable properties of β-TCP. In conclusion, the controlled release of 5 μg G-CSF significantly enhanced the osteoconductive and biodegradable properties of β-TCP. The combination of G-CSF slow-release and β-TCP is a novel and promising approach for treating pediatric craniofacial bone defects.
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Abstract
PURPOSE Endothelial progenitor cells (EPCs) represent a population of novel precursor cells with known ability to participate in angiogenesis. Our previous studies have shown that local EPC therapy significantly increased angiogenesis and osteogenesis to promote fracture healing in an animal bone defect model. However, the cellular and molecular mechanisms by which EPC therapy promotes fracture healing remain largely unknown. The purpose of this study was to quantify local bone morphogenetic protein (BMP-2) expression after EPC therapy for a rat segmental bone defect, in hopes of further defining the potential mechanisms by which EPCs promote fracture healing. METHOD EPCs were isolated from the bone marrow of syngeneic rats and cultured ex vivo for 7-10 days before transfer to the bone defect. A total of 56 rats were studied. The treatment group received 1 × 10 EPCs on a gelfoam scaffold at the bone defect, and control animals received gelfoam/saline only. Before euthanasia, radiographs of the femur were performed. Animals were euthanized at 1, 2, 3, and 10 weeks, and specimens from the fracture gap area were collected, pulverized, and total messenger RNA (mRNA) was extracted. BMP-2 mRNA was measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. All measurements were performed in triplicate. RESULTS All EPC-treated bone defects healed radiographically by 10 weeks, whereas control-treated defects developed a nonunion. The expression of BMP-2 mRNA was significantly elevated in EPC-treated defects relative to controls at week 1 (EPC, 0.59 ± 0.10; control, 0.31 ± 0.08; P = 0.05), week 2 (EPC, 0.40 ± 0.06; control, 0.23 ± 0.04; P = 0.04), and week 3 (EPC, 0.33 ± 0.06; control, 0.18 ± 0.03; P = 0.04), but not at week 10 (EPC, 0.31 ± 0.06; control, 0.21 ± 0.04, P = 0.15). The highest mean expression of BMP-2 in EPC-treated defects was observed at 1 week, with a progressive decline in BMP-2 expression noted thereafter. CONCLUSIONS These findings demonstrate that EPC-treated bone defects demonstrate both radiographic healing and elevated expression of BMP-2 relative to control-treated defects. These results provide further insight into the potential mechanisms by which EPC therapy may promote fracture healing and provide further evidence to suggest that the trophic actions of EPC therapy may be a critical factor in their contribution to fracture healing.
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Kuroda R, Matsumoto T, Kawakami Y, Fukui T, Mifune Y, Kurosaka M. Clinical impact of circulating CD34-positive cells on bone regeneration and healing. TISSUE ENGINEERING PART B-REVIEWS 2014; 20:190-9. [PMID: 24372338 DOI: 10.1089/ten.teb.2013.0511] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Failures in fracture healing after conventional autologous and allogenic bone grafting are mainly due to poor vascularization. To meet the clinical demand, recent attentions in the regeneration and repair of bone have been focused on the use of stem cells such as bone marrow mesenchymal stem cells and circulating skeletal stem cells. Circulating stem cells are currently paid a lot of attention due to their ease of clinical setting and high potential for osteogenesis and angiogenesis. In this report, we focus on the first proof-of-principle experiments demonstrating the collaborative characteristics of circulating CD34(+) cells, known as endothelial and hematopoietic progenitor cell-rich population, which are capable to differentiate into both endothelial cells and osteoblasts. Transplantation of circulating CD34(+) cells provides a favorable environment for fracture healing via angiogenesis/vasculogenesis and osteogenesis, finally leading to functional recovery from fracture. Based on a series of basic studies, we performed a phase 1/2 clinical trial of autologous CD34(+) cell transplantation in patients with tibial or femoral nonunions and reported the safety and efficacy of this novel therapy. In this review, the current concepts and strategies in circulating CD34(+) cell-based therapy and its potential applications for bone repair will be highlighted.
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Affiliation(s)
- Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine , Kobe, Japan
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Kuroda R, Matsumoto T, Niikura T, Kawakami Y, Fukui T, Lee SY, Mifune Y, Kawamata S, Fukushima M, Asahara T, Kawamoto A, Kurosaka M. Local transplantation of granulocyte colony stimulating factor-mobilized CD34+ cells for patients with femoral and tibial nonunion: pilot clinical trial. Stem Cells Transl Med 2014; 3:128-34. [PMID: 24307697 PMCID: PMC3902290 DOI: 10.5966/sctm.2013-0106] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Most bone fractures typically heal, although a significant proportion (5%-10%) of fractures fail to heal, resulting in delayed union or persistent nonunion. Some preclinical evidence shows the therapeutic potential of peripheral blood CD34(+) cells, a hematopoietic/endothelial progenitor cell-enriched population, for bone fracture healing; however, clinical outcome following transplantation of CD34(+) cells in patients with fracture has never been reported. We report a phase I/IIa clinical trial regarding transplantation of autologous, granulocyte colony stimulating factor-mobilized CD34(+) cells with atelocollagen scaffold for patients with femoral or tibial fracture nonunion (n = 7). The primary endpoint of this study is radiological fracture healing (union) by evaluating anteroposterior and lateral views at week 12 following cell therapy. For the safety evaluation, incidence, severity, and outcome of all adverse events were recorded. Radiological fracture healing at week 12 was achieved in five of seven cases (71.4%), which was greater than the threshold (18.1%) predefined by the historical outcome of the standard of care. The interval between cell transplantation and union, the secondary endpoint, was 12.6 ± 5.4 weeks (range, 8-24 weeks) for clinical healing and 16.1 ± 10.2 weeks (range, 8-36 weeks) for radiological healing. Neither deaths nor life-threatening adverse events were observed during the 1-year follow-up after the cell therapy. These results suggest feasibility, safety, and potential effectiveness of CD34(+) cell therapy in patients with nonunion.
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Seo SG, Yeo JH, Kim JH, Kim JB, Cho TJ, Lee DY. Negative-pressure wound therapy induces endothelial progenitor cell mobilization in diabetic patients with foot infection or skin defects. Exp Mol Med 2013; 45:e62. [PMID: 24232261 PMCID: PMC3849576 DOI: 10.1038/emm.2013.129] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 08/17/2013] [Accepted: 09/02/2013] [Indexed: 02/06/2023] Open
Abstract
Non healing chronic wounds are difficult to treat in patients with diabetes and can result in severe medical problems for these patients and for society. Negative-pressure wound therapy (NPWT) has been adopted to treat intractable chronic wounds and has been reported to be effective. However, the mechanisms underlying the effects of this treatment have not been elucidated. To assess the vasculogenic effect of NPWT, we evaluated the systemic mobilization of endothelial progenitor cells (EPCs) during NPWT. Twenty-two of 29 consecutive patients who presented at the clinic of Seoul National Universty Hospital between December 2009 and November 2010 who underwent NPWT for diabetic foot infections or skin ulcers were included in this study. Peripheral blood samples were taken before NPWT (pre-NPWT) and 7–14 days after the initiation of NPWT (during-NPWT). Fluorescence-activated cell sorting (FACS) analysis showed that the number of cells in EPC-enriched fractions increased after NPWT, and the numbers of EPC colony forming units (CFUs) significantly increased during NPWT. We believe that NPWT is useful for treating patients with diabetic foot infections and skin ulcers, especially when these conditions are accompanied by peripheral arterial insufficiency. The systemic mobilization of EPCs during NPWT may be a mechanism for healing intractable wounds in diabetic patients with foot infections or skin defects via the formation of increased granulation tissue with numerous small blood vessels.
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Affiliation(s)
- Sang Gyo Seo
- Department of Orthopedic Surgery, Seoul National University College of Medicine, Seoul, Korea
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Coculture of peripheral blood CD34+ cell and mesenchymal stem cell sheets increase the formation of bone in calvarial critical-size defects in rabbits. Br J Oral Maxillofac Surg 2013; 52:134-9. [PMID: 24210781 DOI: 10.1016/j.bjoms.2013.10.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2013] [Accepted: 10/11/2013] [Indexed: 01/27/2023]
Abstract
The reconstruction of large bony defects remains a clinical challenge, and angiogenesis and neovascularisation are being given more attention in bone tissue engineering. In this study we cocultured peripheral blood CD34+ cells (PB-CD34+ cells), an endothelial progenitor cell/haematopoietic stem cell-enriched population, with bone marrow-derived mesenchymal stem cells (MSC) to investigate their potential for bony regeneration. Cocultured cells showed better osteogenic differentiation than MSC alone in vitro. The cocultured cells and MSC sheets were also composited with hydroxyapatite and implanted in calvarial critical-size defects in rabbits. The rabbits were killed before microcomputed tomographic (MicroCT) and histological analysis. The results showed that cocultured cell composites had promoted bony regeneration more efficiently by 8 weeks after implantation. Our results indicate that the coculture of PB-CD34+ cells and MSC increases bony regeneration in calvarial critical-size defects in rabbits, and provide a new promising therapeutic strategy to aid skeletal healing.
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Toupadakis CA, Granick JL, Sagy M, Wong A, Ghassemi E, Chung DJ, Borjesson DL, Yellowley CE. Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model. Cytotherapy 2013; 15:1136-47. [PMID: 23831362 DOI: 10.1016/j.jcyt.2013.05.004] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2012] [Revised: 03/26/2013] [Accepted: 05/08/2013] [Indexed: 12/16/2022]
Abstract
BACKGROUND AIMS Delivery of bone marrow-derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. METHODS A transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro-computed tomography. RESULTS AMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. CONCLUSIONS Our data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.
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Affiliation(s)
- Chrisoula A Toupadakis
- Department of Anatomy, Physiology & Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, USA
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Marmotti A, Castoldi F, Rossi R, Marenco S, Risso A, Ruella M, Tron A, Borrè A, Blonna D, Tarella C. Bone marrow-derived cell mobilization by G-CSF to enhance osseointegration of bone substitute in high tibial osteotomy. Knee Surg Sports Traumatol Arthrosc 2013; 21:237-48. [PMID: 22872005 DOI: 10.1007/s00167-012-2150-z] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2012] [Accepted: 07/19/2012] [Indexed: 12/14/2022]
Abstract
PURPOSE To evaluate granulocyte colony-stimulating factor (G-CSF) efficacy in accelerating bone regeneration following opening-wedge high tibial valgus osteotomy for genu varum. METHODS A phase II trial was conducted for evaluating the preoperative administration of G-CSF given at 10 μg/kg/day for 3 consecutive days with an additional half-dose 4 h before the opening-wedge high tibial valgus osteotomy. Overall, 12 patients (Group A) received G-CSF treatment, and the subsequent 12 patients (Group B) underwent surgery without G-CSF. The osteotomy gap was filled by a bone graft substitute. Bone marrow cell (BMC) mobilization was monitored by CD34+ve cell and clonogenic progenitor cell analysis. All patients underwent a clinical (Lysholm Knee Scale and SF-36) and radiographic evaluation preoperatively, as well as at given intervals postsurgery. RESULTS All patients completed the treatment program without major side effects; G-CSF was well tolerated. BMC mobilization occurred in all Group A patients, with median peak values of circulating CD34+ve cells of 110/μL (range 29-256). Circulating clonogenic progenitors paralleled CD34+ve cell levels. A significant improvement in Lysholm Knee Scale was recorded at follow-up in Group A compared to Group B. At the radiographic evaluation, there was a significant increase in osseointegration at the bone-graft junction in Group A at 1, 2, 3 and 6 months postsurgery compared to Group B. The computerized tomography scan of the grafted area at 2 months postsurgery showed no significant difference in the quality of the newly formed bone between the two Groups. CONCLUSIONS Although the limited number of patients does not allow firm conclusions, the study suggests that G-CSF can be safely administered preoperatively in subjects undergoing opening-wedge high tibial valgus osteotomy; in addition, the clinical, radiographic and CT monitoring indicate that G-CSF and/or mobilized BMCs may hasten bone graft substitute osseointegration. LEVEL OF EVIDENCE I.
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Affiliation(s)
- A Marmotti
- Department of Orthopaedics and Traumatology, Ordine Mauriziano, Umberto I Hospital, University of Torino, Turin, Italy.
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Kawakami Y, Ii M, Alev C, Kawamoto A, Matsumoto T, Kuroda R, Shoji T, Fukui T, Masuda H, Akimaru H, Mifune Y, Kuroda T, Horii M, Yokoyama A, Kurosaka M, Asahara T. Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing. Cell Transplant 2012; 21:2689-709. [DOI: 10.3727/096368912x654920] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Transplantation of bone marrow (BM) CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34+ cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34+ cell expansion method. Seven-day ex vivo expansion culture of BM CD34+ cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34+ cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34+ cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34+ cells and fresh BM CD34+ cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34+ cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34+ cells. In vitro, cEx-BM CD34+ cells showed higher colony/tube-forming capacity than nonexpanded BM CD34+ cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34+ cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34+ cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.
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Affiliation(s)
- Yohei Kawakami
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Masaaki Ii
- Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan
| | - Cantas Alev
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan
| | - Atsuhiko Kawamoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Tomoyuki Matsumoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Taro Shoji
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoaki Fukui
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Haruchika Masuda
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
| | - Hiroshi Akimaru
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Yutaka Mifune
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoya Kuroda
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Miki Horii
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Ayumi Yokoyama
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Takayuki Asahara
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
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Abstract
OBJECTIVES Angiogenesis and osteogenesis are essential for bone growth, fracture repair, and bone remodeling. Vascular endothelial growth factor (VEGF) has an important role in bone repair by promoting angiogenesis and osteogenesis. In our previous study, endothelial progenitor cells (EPCs) promoted bone healing in a rat segmental bone defect as confirmed by radiological, histological, biomechanical, and micro-CT evaluations. Although EPCs have demonstrated effectiveness in animal models of fracture healing, the mechanism by which EPCs enhance fracture healing remains unclear. We hypothesized a possible paracrine mechanism of action, where the secretion of growth factors critical to the processes of fracture healing (such as VEGF), is responsible for the positive effects of EPC therapy. The purpose of this study was to evaluate VEGF gene expression after local EPC therapy for a rat segmental bone defect. METHODS Rat bone marrow-derived EPCs were isolated by the Ficoll-paque gradient centrifuge technique. The EPCs were cultured for 7-10 days in endothelial cell growth medium with supplements and collected for treatment of the rat segmental bone defect. EPCs were identified by immunocytochemistry staining with primary antibodies for CD34, CD133, fetal liver kinase-1, and Von Willebrand factor. A total of 56 rats were studied. A 5-mL segmental bone defect was created in the middle one-third of each femur followed by miniplate fixation. The treatment group received 1 × 10 EPCs locally at the bone defect on a gelfoam scaffold and control animals received the gelfoam scaffold only. Seven control and 7 EPC-treated rats were included in each group at 1, 2, 3, and 10 weeks. The animals were sacrificed at the end of the treatment period, and specimens from the fracture gap area were collected and immediately frozen. Rat VEGF mRNA was measured by reverse-transcriptase-polymerase chain reaction and quantified by VisionWorksLS. All measurements were performed in triplicate. RESULTS Cultured EPCs at 1 week showed positive staining for CD34, CD133, fetal liver kinase-1, and Von Willebrand factor markers. The EPC group had a greater VEGF expression than the control group at weeks 1, 2, and 3, but not at week 10. Three VEGF isoforms were detected in this rat model: VEGF120, VEGF164, and VEGF188. VEGF120 and VEGF164 levels peaked at 2 weeks, whereas VEGF188 levels peaked at 3 weeks. All 3 VEGF isoform levels were low at 10 weeks. DISCUSSION AND CONCLUSIONS EPC-based therapy for a segmental bone defect results in increased VEGF expression during the early period of fracture repair. In addition, the specific VEGF isoform may be a key regulator of the bone healing process. These findings demonstrate that EPCs may promote fracture healing by increasing VEGF levels and thus stimulating angiogenesis, a process that is essential for early callus formation and bone regeneration.
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Liu Y, Chan JKY, Teoh SH. Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems. J Tissue Eng Regen Med 2012; 9:85-105. [DOI: 10.1002/term.1617] [Citation(s) in RCA: 117] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Revised: 07/13/2012] [Accepted: 08/25/2012] [Indexed: 12/16/2022]
Affiliation(s)
- Yuchun Liu
- Division of Bioengineering, School of Chemical and Biomedical Engineering; Nanyang Technological University; Singapore 637459
- Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine; National University of Singapore; Singapore 119228
| | - Jerry K Y Chan
- Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine; National University of Singapore; Singapore 119228
- Department of Reproductive Medicine, KK Women's and Children's Hospital; Singapore 229899
- Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School; Singapore
| | - Swee-Hin Teoh
- Division of Bioengineering, School of Chemical and Biomedical Engineering; Nanyang Technological University; Singapore 637459
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Long-term clinical outcome after intramuscular transplantation of granulocyte colony stimulating factor-mobilized CD34 positive cells in patients with critical limb ischemia. Atherosclerosis 2012; 224:440-5. [DOI: 10.1016/j.atherosclerosis.2012.07.031] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Revised: 07/17/2012] [Accepted: 07/18/2012] [Indexed: 01/24/2023]
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Fadini GP, Rattazzi M, Matsumoto T, Asahara T, Khosla S. Emerging role of circulating calcifying cells in the bone-vascular axis. Circulation 2012; 125:2772-81. [PMID: 22665885 DOI: 10.1161/circulationaha.112.090860] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Mehrotra M, Williams CR, Ogawa M, LaRue AC. Hematopoietic stem cells give rise to osteo-chondrogenic cells. Blood Cells Mol Dis 2012; 50:41-9. [PMID: 22954476 DOI: 10.1016/j.bcmd.2012.08.003] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2012] [Revised: 08/08/2012] [Accepted: 08/08/2012] [Indexed: 12/15/2022]
Abstract
Repair of bone fracture requires recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts. Given the close association of bone and bone marrow (BM), it has been suggested that BM may serve as a source of these progenitors. To test the ability of hematopoietic stem cells (HSCs) to give rise to osteo-chondrogenic cells, we used a single HSC transplantation paradigm in uninjured bone and in conjunction with a tibial fracture model. Mice were lethally irradiated and transplanted with a clonal population of cells derived from a single enhanced green fluorescent protein positive (eGFP+) HSC. Analysis of paraffin sections from these animals showed the presence of eGFP+ osteocytes and hypertrophic chondrocytes. To determine the contribution of HSC-derived cells to fracture repair, non-stabilized tibial fracture was created. Paraffin sections were examined at 7 days, 2 weeks and 2 months after fracture and eGFP+ hypertrophic chondrocytes, osteoblasts and osteocytes were identified at the callus site. These cells stained positive for Runx-2 or osteocalcin and also stained for eGFP demonstrating their origin from the HSC. Together, these findings strongly support the concept that HSCs generate bone cells and suggest therapeutic potentials of HSCs in fracture repair.
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Affiliation(s)
- Meenal Mehrotra
- Department of Veterans Affairs Medical Center, Ralph H. Johnson VAMC, Medical University of South Carolina, Charleston, SC, USA
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Ravichandran R, Venugopal JR, Sundarrajan S, Mukherjee S, Sridhar R, Ramakrishna S. Composite poly-l-lactic acid/poly-(α,β)-dl-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2012; 32:1443-51. [DOI: 10.1016/j.msec.2012.04.024] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/08/2011] [Revised: 02/20/2012] [Accepted: 04/19/2012] [Indexed: 01/22/2023]
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Aggarwal R, Lu J, Kanji S, Joseph M, Das M, Noble GJ, McMichael BK, Agarwal S, Hart RT, Sun Z, Lee BS, Rosol TJ, Jackson R, Mao HQ, Pompili VJ, Das H. Human umbilical cord blood-derived CD34+ cells reverse osteoporosis in NOD/SCID mice by altering osteoblastic and osteoclastic activities. PLoS One 2012; 7:e39365. [PMID: 22724005 PMCID: PMC3377665 DOI: 10.1371/journal.pone.0039365] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2012] [Accepted: 05/23/2012] [Indexed: 12/18/2022] Open
Abstract
Background Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Methods and Findings The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality. Conclusions These findings demonstrate a novel approach utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis.
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Affiliation(s)
- Reeva Aggarwal
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Jingwei Lu
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Suman Kanji
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Matthew Joseph
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Manjusri Das
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Garrett J. Noble
- Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, Ohio, United States of America
| | - Brooke K. McMichael
- Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America
| | - Sudha Agarwal
- Division of Oral Biology, Department of Orthopedics, College of Dentistry, The Ohio State University, Columbus, Ohio, United States of America
| | - Richard T. Hart
- Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, Ohio, United States of America
| | - Zongyang Sun
- Division of Oral Biology, Department of Orthopedics, College of Dentistry, The Ohio State University, Columbus, Ohio, United States of America
| | - Beth S. Lee
- Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America
| | - Thomas J. Rosol
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, United States of America
| | - Rebecca Jackson
- Division of Endocrinology, Diabetes and Metabolism, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America
| | - Hai-Quan Mao
- Department of Materials Science and Engineering, John's Hopkins University, Baltimore, Maryland, United States of America
| | - Vincent J. Pompili
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
| | - Hiranmoy Das
- Cardiovascular Stem Cell Research Laboratory, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, United States of America
- * E-mail:
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Fukui T, Ii M, Shoji T, Matsumoto T, Mifune Y, Kawakami Y, Akimaru H, Kawamoto A, Kuroda T, Saito T, Tabata Y, Kuroda R, Kurosaka M, Asahara T. Therapeutic effect of local administration of low-dose simvastatin-conjugated gelatin hydrogel for fracture healing. J Bone Miner Res 2012; 27:1118-31. [PMID: 22275312 DOI: 10.1002/jbmr.1558] [Citation(s) in RCA: 68] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Several reports have shown the therapeutic effect of statins on bone formation and neovascularization. However, the effect of the systemic administration of statins is limited due to its metabolism in the liver and clearance in the digestive system. In addition, high-dose administration may cause adverse side effects. To avoid low-efficacy/frequent side effects of high-dose statin treatment, we utilized biodegradable gelatin hydrogel as a drug delivery system of statin for fracture healing. A femoral fracture was created in rats with periosteum cauterization leading to nonunion at 8 weeks postfracture. Rats received local administration of either simvastatin-conjugated gelatin hydrogel (ST-Gel group) or gelatin hydrogel alone (Gel group). Approximately 70% of animals in the ST-Gel group achieved fracture union radiographically and histologically, while only 7% of animals achieved fracture healing in the Gel group. Functional bone healing was also significantly greater with increased angiogenesis- and osteogenesis-related growth factor expressions in periosteal granulation tissue in the ST-Gel group than in the Gel group. Simvastatin locally applied with gelatin hydrogel to fracture sites at a dose similar to that used in clinical settings successfully induced fracture union in a rat unhealing bone fracture model via its effect on both angiogenesis and osteogenesis.
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Affiliation(s)
- Tomoaki Fukui
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Japan
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Fukui T, Matsumoto T, Mifune Y, Shoji T, Kuroda T, Kawakami Y, Kawamoto A, Ii M, Kawamata S, Kurosaka M, Asahara T, Kuroda R. Local Transplantation of Granulocyte Colony-Stimulating Factor-Mobilized Human Peripheral Blood Mononuclear Cells for Unhealing Bone Fractures. Cell Transplant 2012. [DOI: 10.3727/096368911x582769a] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
We previously reported the therapeutic potential of human peripheral blood (hPB) CD34+ cells for bone fracture healing via vasculogenesis/angiogenesis and osteogenesis. Transplantation of not only hPB CD34+ cells but also hPB total mononuclear cells (MNCs) has shown their therapeutic efficiency for enhancing ischemic neovascularization. Compared with transplantation of purified hPB CD34+ cells, transplantation of hPB MNCs is more attractive due to its simple method of cell isolation and inexpensive cost performance in the clinical setting. Thus, in this report, we attempted to test a hypothesis that granulocyte colony-stimulating factor-mobilized (GM) hPB MNC transplantation could also contribute to fracture healing via vasculogenesis/angiogenesis and osteogenesis. Nude rats with unhealing fractures received local administration of the following materials with atelocollagen: 1 × 107 GM hPB MNCs (Hi group), 1 × 106 GM hPB MNCs (Lo group), or PBS (PBS group). Immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated human cell-derived vasculogenesis and osteogenesis in the Hi and Lo groups, but not in the PBS group at week 1. Intrinsic angiogenesis and osteogenesis assessed by rat capillary, osteoblast density, and real-time RT-PCR analysis was significantly enhanced in the Hi group compared to the other groups. Blood flow assessment by laser doppler perfusion imaging showed a significantly higher blood flow ratio at week 1 in the Hi group compared with the other groups. Morphological fracture healing was radiographically and histologically confirmed in about 30% of animals in the Hi group at week 8, whereas all animals in the other groups resulted in nonunion. Local transplantation of GM hPB MNCs contributes to fracture healing via vasculogenesis/angiogenesis and osteogenesis.
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Affiliation(s)
- Tomoaki Fukui
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoyuki Matsumoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Yutaka Mifune
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Taro Shoji
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoya Kuroda
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Yohei Kawakami
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Atsuhiko Kawamoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Masaaki Ii
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Shin Kawamata
- Stem Cell Bank Research Group, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Takayuki Asahara
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
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Affiliation(s)
- Aaron Nauth
- Department of Surgery, Division of Orthopaedics, St. Michael's Hospital, University of Toronto, Toronto, Canada
| | - Emil H Schemitsch
- Department of Surgery, Division of Orthopaedics, St. Michael's Hospital, University of Toronto, Toronto, Canada,Address for correspondence: Dr. Emil H. Schemitsch, St. Michael's Orthopaedic Associates, 55 Queen Street East, Suite 800, Toronto, Ontario, Canada M5C 1R6. E-mail:
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Undale A, Fraser D, Hefferan T, Kopher RA, Herrick J, Evans GL, Li X, Kakar S, Hayes M, Atkinson E, Yaszemski MJ, Kaufman DS, Westendorf JJ, Khosla S. Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow. J Orthop Res 2011; 29:1804-11. [PMID: 21674605 PMCID: PMC3179810 DOI: 10.1002/jor.21480] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2011] [Accepted: 05/23/2011] [Indexed: 02/04/2023]
Abstract
Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10-12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.
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Affiliation(s)
- Anita Undale
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Daniel Fraser
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Theresa Hefferan
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Ross A. Kopher
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - James Herrick
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Glenda L. Evans
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Xiaodong Li
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Sanjeev Kakar
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Meredith Hayes
- Orthopedic Research, College of Medicine, Mayo Clinic, Rochester, MN, USA
| | | | | | - Dan S. Kaufman
- Department of Medicine and Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Sundeep Khosla
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, MN, USA
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Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis. Plast Reconstr Surg 2011; 128:395-405. [PMID: 21788831 DOI: 10.1097/prs.0b013e31821e6e10] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Although bone repair is a relatively efficient process, a significant portion of patients fail to heal their fractures. Because adequate blood supply is essential to osteogenesis, the authors hypothesize that augmenting neovascularization by increasing the number of circulating progenitor cells will improve bony healing. METHODS Bilateral full-thickness defects were created in the parietal bones of C57 wild-type mice. Intraperitoneal AMD3100 (n = 33) or sterile saline (n = 33) was administered daily beginning on postoperative day 3 and continuing through day 18. Circulating progenitor cell number was quantified by fluorescence-activated cell sorting. Bone regeneration was assessed with micro-computed tomography. Immunofluorescent CD31 and osteocalcin staining was performed to assess for vascularity and osteoblast density. RESULTS AMD3100 treatment increased circulating progenitor cell levels and significantly improved bone regeneration. Calvarial defects of AMD3100-treated mice demonstrated increased vascularity and osteoblast density. CONCLUSIONS Improved bone regeneration in this model was associated with elevated circulating progenitor cell number and subsequently improved neovascularization and osteogenesis. These findings highlight the importance of circulating progenitor cells in bone healing and may provide a novel therapy for bone regeneration.
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Matsumoto T, Ingham SM, Mifune Y, Osawa A, Logar A, Usas A, Kuroda R, Kurosaka M, Fu FH, Huard J. Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells. Stem Cells Dev 2011; 21:859-72. [PMID: 21732814 DOI: 10.1089/scd.2010.0528] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.
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Affiliation(s)
- Tomoyuki Matsumoto
- Stem Cell Research Center, Children's Hospital of Pittsburgh, and Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA
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Alev C, Ii M, Asahara T. Endothelial progenitor cells: a novel tool for the therapy of ischemic diseases. Antioxid Redox Signal 2011; 15:949-65. [PMID: 21254837 DOI: 10.1089/ars.2010.3872] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Circulating endothelial progenitor cells (EPCs) are believed to home to sites of neovascularization, contributing to vascular regeneration either directly via incorporation into newly forming vascular structures or indirectly via the secretion of pro-angiogenic growth factors, thereby enhancing the overall vascular and hemodynamic recovery of ischemic tissues. The therapeutic application of EPCs has been shown to be effective in animal models of ischemia, and we as well as other groups involved in clinical trials have demonstrated that the use of EPCs was safe and feasible for the treatment of critical limb ischemia and cardiovascular diseases. However, many issues in the field of EPC biology, especially in regard to the proper and unambiguous molecular characterization of these cells, still remain unresolved, hampering not only basic research but also the effective therapeutic use and widespread application of these cells. Further, recent evidence suggests that several diseases and pathological conditions are correlated with a reduction in the number and biological activity of EPCs, making the development of novel strategies to overcome the current limitations and shortcomings of this promising but still limited therapeutic tool by refinement and improvement of EPC purification, expansion, and administration techniques, a rather pressing issue.
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Affiliation(s)
- Cantas Alev
- Group of Vascular Regeneration Research, Institute of Biomedical Research and Innovation/RIKEN Center for Developmental Biology, Kobe, Japan
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Khosla S, Westendorf JJ, Mödder UI. Concise review: Insights from normal bone remodeling and stem cell-based therapies for bone repair. Stem Cells 2011; 28:2124-8. [PMID: 20960512 DOI: 10.1002/stem.546] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
There is growing interest in the use of mesenchymal stem cells for bone repair. As a major reason for normal bone remodeling is the removal of fatigue microcracks, advances in our understanding of this process may inform approaches to enhance fracture healing. Increasing evidence now indicates that physiological bone remodeling occurs in close proximity to blood vessels and that these vessels carry perivascular stem cells that differentiate into osteoblasts. Similarly, fracture healing is critically dependent on the ingrowth of blood vessels not only for a nutrient supply but also for the influx of osteoblasts. A number of animal and human studies have now shown the potential benefit of bone marrow-derived mesenchymal stem cells in enhancing bone repair. However, as in other tissues, the question of whether these cells improve fracture healing directly by differentiating into osteoblasts or indirectly by secreting paracrine factors that recruit blood vessels and the accompanying perivascular stem cells remains a major unresolved issue. Moreover, CD34+ cells, which are enriched for endothelial/hematopoietic cells, have also shown efficacy in various bone repair models, at least in part due to the induction of angiogenesis and recruitment of host progenitor cells. Thus, mesenchymal and nonmesenchymal stem/progenitor cells are attractive options for bone repair. It is possible that they contribute directly to bone repair, but it is also likely that they express paracrine factors in the appropriate amounts and combinations that promote and sustain the healing process.
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Affiliation(s)
- Sundeep Khosla
- Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
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New method to produce hemocomponents for regenerative use from peripheral blood: integration among platelet growth factors monocytes and stem cells. Transfus Apher Sci 2011; 42:117-24. [PMID: 20227343 DOI: 10.1016/j.transci.2010.01.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Recent studies have shown the importance of monocytes/macrophageses and of CD34+ progenitors in tissue regeneration processes. These cells, obtained generally from bone marrow, are seen in damaged tissue. We have studied a method to collect from the peripheral blood, using a cell separator and without stimulation of the patient/donor, a leukocyte platelet concentrated hemocomponent (CLP) for regenerative use which contains platelets, monocytes/macrophages, fibrinogen and CD34+ cells. We appraised the composition and cell functionality of the final hemocomponent during production and cryoconservation. The results show a positive increase in concentration values, in comparison with the pre-collection, of the cells that were involved in regeneration; i.e. the platelets, monocytes and CD34+ cells. These concentrations were also maintained at an effective level during cryoconservation of the hemocomponent. The CLP also demonstrated positive clonogenic potential in culture, showing that the CD34+ progenitors involved in CFU formation are functional in the fresh and thawed product. In brief we have shown that it is possible to produce, in a simple way, a hemocomponent for regenerative use that is standardized, reliable, and is economically feasible.
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50
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Ishida K, Matsumoto T, Sasaki K, Mifune Y, Tei K, Kubo S, Matsushita T, Takayama K, Akisue T, Tabata Y, Kurosaka M, Kuroda R. Bone regeneration properties of granulocyte colony-stimulating factor via neovascularization and osteogenesis. Tissue Eng Part A 2011; 16:3271-84. [PMID: 20626235 DOI: 10.1089/ten.tea.2009.0268] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
OBJECTIVES It has been well recognized that appropriate vascularization is emerging as a prerequisite for bone development and regeneration. The aim of this study was to test the hypothesis that locally applied granulocyte colony-stimulating factor (G-CSF) enhances bone regeneration via revascularization and osteogenesis. METHODS A segmental bone defect (20mm) was created at the diaphysis of the rabbit ulna. The defects were treated with cationized gelatin hydrogel, which was the drug delivery system, with G-CSF, and then bone regeneration, neovascularization, and osteogenesis properties with G-CSF were assessed. RESULTS Radiographic, computed tomography, and histological findings revealed that bone formation was significantly promoted in G-CSF-treated group as early as 2 weeks. Immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and flow cytometry studies indicated that angiogenesis/vasculogenesis, which are regulated by mobilization and incorporation of CD34+/G-CSF receptor (CSFR+) cells, and osteogenesis, which is regulated by osteocalcin+/G-CSFR+ cells, were also significantly enhanced in the G-CSF group. CONCLUSIONS This study suggests that locally applied G-CSF contributes to an ideal local environment for fracture healing by supplying adequate blood flow and stimulating osteogenesis. G-CSF may have the therapeutic potential for bone regeneration.
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Affiliation(s)
- Kazunari Ishida
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
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