2006 Fiscal Year Final Research Report Summary
Ligament and meniscus regeneration using cells in peripheral blood
| Project/Area Number |
17591571
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Kobe University |
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Principal Investigator |
KURODA Ryosuke Kobe University, University Hospital, Lecturer, 医学部附属病院, 講師 (80379362)
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| Co-Investigator(Kenkyū-buntansha) |
KUROSAKA Masahiro Kobe University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (70170115)
YOSHIYA Shinichi Hyogo College of Medicine, Orthopedic surgery, Professor, 整形外科, 教授 (00201070)
MURATSU Hirotsugu Kobe University, Graduate School of Medicine, Visiting Medical Scientist, 大学院・医学系研究科, 医学研究員 (30273783)
FUJIOKA Hiroyuki Kobe University, Graduate School of Medicine, Lecturer, 大学院・医学系研究科, 講師 (10252777)
MIZUNO Kiyonori Kobe University, Graduate School of Medicine, Lecturer, 大学院・医学系研究科, 特命講師 (40418778)
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| Project Period (FY) |
2005 – 2006
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| Keywords | Regenerative medicine / Peripheral blood stem cell / Platelet-rich plasma / Meniscus / Ligament / Orthopaedic surgery |
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| Research Abstract |
1. Platelet-Rich Plasma
The objective of the study was to test the hypothesis that platelet-rich plasma (PRP) enhances meniscaltissue regeneration in vitro and in vivo. In the in vitro study, monolayer meniscal cell cultures were prepared, and 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay and 5-bromo-20-deoxyuridine assay were performed to assess proliferative behavior in the presence of PRP. Alcian blue assay was performed to assess extracellular matrix (ECM) synthesis. To detect the fibrocartilage-related messenger ribonucleic acid (mRNA) expressions, real-time polymerase chain reaction was performed. In the in vivo study, 1.5-mm-diameter full-thickness defects were created in the avascular region of rabbit meniscus. Gelatin hydrogel (GH) was used as the drug delivery system for PRP growth factors. The defects were filled as follows : Group A, GH with PRP ; Group B, GH with platelet-poor plasma ; Group C, GH only. Each group w
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as evaluated histologically at 4, 8, and 12 weeks after surgery. PRP stimulated deoxyribonucleic acid synthesis and ECM synthesis ( p < 0.05). Meniscal cells cultured with PRP showed greater mRNA expression of biglycan and decorin ( p < 0.05). Histological findings showed that remnants of gelatin hydrogels existed at 4 weeks, indicating that the hydrogels could control release for approximately 4 weeks. Histological scoring of the defect sites at 12 weeks revealed significantly better meniscal repair in animals that received PRP with GH than in the other two groups. These findings suggest that PRP enhances the healing of meniscal defects.
2. Peripheral Blood Stem Cells (Human Circulating CD34+ Cells)
Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to contribute to neoangiogenesis, however the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte-stimulating factor mobilized peripheral blood CD34+ cells with atelocollagen (CD34+ group), or only atelocollagen (control group) were locally transplanted after creating medial collateral ligament injury in immunodeficient rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining at the injury site demonstrated molecular and histological expression of human-specific markers for endothelial cells in the CD34+ group at week 1. Endogeneous effect assessed by capillary density and mRNA expression of vascular endothelial growth factor was found by CD34+ cell transplantation. Gene expression of ligament-specific marker in the CD34+ group assessed by real time RT'PCR was significant higher than in the control group. Ligament healing assessed by macroscopic and histological examinations was enhanced by CD34+ cell transplantation. Our data suggest circulating human CD34+ cells may play an essential role in the ligament healing process by promoting a favorable environment through neovascularization in the damaged tissue, resulting in appropriate ligament healing. Less
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Research Products
(4 results)
