목차

Ⅰ. INTRODUCTION 1
1. Pathophysiology of spinal cord injury 1
2. Intrinsic factors for axonal regeneration following SCI 2
3. Extrinsic factors for axonal regeneration following SCI 3
3.1. Glial scar 3
3.2. Fibrotic scar 5
3.3. Chondroitin sulfate proteoglycans (CSPGs) 7
3.4. Fluid-filled cystic cavities 7
4. Injectable hydrogel for spinal cord repair 9
5. Cell transplantation therapy for spinal cord repair 11
6. Aim of this study 13
Ⅱ. MATERIALS AND METHODS 15
1. Animals and surgical procedures 15
2. Preparation of I-5 or I-5/ARSB complex 15
3. Tissue processing and histological assessment 16
4. Quantification of axonal growth 19
5. Corticospinal and propriospinal tract axon tracing 20
6. Behavior assessment of locomotor recovery 21
7. In vitro fibrotic scar model 22
8. Western blot analysis 23
9. Glygosaminoglycan assay 24
10. in vivo sulfatase assay 24
11. NSCs preparation 25
12. NSCs transplantation 25
13. NSCs culture on different stiffness of hydrogel plate in vitro 26
14. Quantification of cell spreading area 26
15. Live/dead cell survival assay 27
16. Quantitative assessment of mRNA expression by real-time PCR 27
17. Pharmacological inhibition of mechanosensitive ion channels 28
18. Assessment of spontaneous calcium activity 28
19. Statistical analysis 29
Ⅲ. RESULTS 30
Part A. Limitation of axon growth into the hydrogel-created ECM following SCI 30
1. Cystic cavity formation initiates at 7 days after SCI 30
2. Injection of the I-5 hydrogel prevents cavity formation 32
3. Limitation of regenerating axons into hydrogel-created ECM 35
Part B. Alleviation of the excessive fibrotic microenvironment in the hydrogel-induced ECM by I-5/ARSB complex 40
1. Characterization of the I-5 hydrogel-induced fibrotic microenvironment at the lesioned spinal cord 40
2. CSPGs stimulate fibrotic matrix production and cellular clustering in an in vitro fibrotic scar model 45
3. Injection of the I-5/ARSB complex reduces deposition of CSPGs and improves locomotor recovery 50
4. Injection of the I-5/ARSB complex alleviates the fibrotic microenvironment in the injured spinal cord 61
5. I-5/ARSB complex induces regeneration of serotonergic axons 67
6. I-5/ARSB complex promotes regeneration of the propriospinal axons into and beyond the hydrogel-created ECM 72
Part C. Modulation of hydrogel mechanical stiffness to improve survival of NSC grafts 76
1. Poor survival of NSC grafts transplanted with I-5 hydrogel 76
2. Influence of substrate stiffness on NSCs behavior in vitro 78
3. Expression of mechanosensitive ion channels in NSCs 84
4. The effect of pharmacological inhibition of mechanosensitive ion channel on NSCs 90
5. High percentage of stiff hydrogel significantly enhanced NSC grafts survival in the lesioned spinal cord 93
Ⅳ. DISCUSSION 98
Ⅴ. SUMMARY AND CONCLUSION 106
REFERENCE 107
국문요약 130