Title Page
Contents
Abstract 9
Chapter 1. Reduction in TNF-α induced Inflammation by Exosome-Mimetic Nanovesicles in Endothelial Cells 11
I. INTRODUCTION 11
II. MATERIALS AND METHODS 14
1.2.1. Preparation of NVs and FT/NVs 14
1.2.2. Characterization of NVs and FT/NVs 14
1.2.3. Cell culture 15
1.2.4. Leukocyte adhesion assay 15
1.2.5. Quantitative real-time PCR 16
1.2.6. Western blot analysis 16
1.2.7. Measurement of reactive oxygen species (ROS) 17
1.2.8. Immunocytochemistry 17
III. RESULTS 19
1.3.1. Efficient production of exosome-mimetic nanovesicles (NVs) by freeze and thaw process 19
1.3.2. NVs inhibit TNF-α-induced leukocyte adhesion and cell adhesion molecule expression in HCAECs 23
1.3.3. NVs decrease the production of proinflammatory cytokines caused by TNF-α. 25
1.3.4. NVs inhibit the oxidative stress by suppressing p38 MAPK pathway induced by TNF-α. 27
1.3.5. NVs inhibited nuclear translocation of NF-κB induced by TNF-α. 30
IV. DISCUSSION 32
V. CONCLUSION 34
Chapter 2. A Biomimetic Scaffold Integrating Human Cell-derived Extracellular Matrix Deposited under Macromolecular Crowding Condition and Pre-osteoblast derived Exosome for Bone Regeneration 35
I. INTRODUCTION 35
II. MATERIALS AND METHODS 39
2.2.1. Cell culture 39
2.2.2. Characterization of MMC agents 39
2.2.3. Cell viability assay 40
2.2.4. Decellularization of recellularization 41
2.2.5. Collagen extraction and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 41
2.2.6. Immunocytochemistry 42
2.2.7. Western blot analysis 43
2.2.8. Preparation and characterization of extracellular vesicle 43
2.2.9. ALP and Alizarin Res S (ARS) staining 44
2.2.10. Statistical Analysis 45
III. RESULTS 46
2.3.1. Biophysical characterization of fucoidan 46
2.3.2. Enhancement of ECM deposition by fucoidan in human dermal fibroblasts 48
2.3.3. Decellularization of accumulated ECM by fucoidan 52
2.3.4. Characterization of pre-osteoblast derived EVs 56
2.3.5. Effect of fucoidan-treated decelluarized ECM and pre-osteoblast-derived EVs on osteogenic differentiation 58
IV. DISCUSSION 61
V. CONCLUSION 64
REFERENCES 65
ABSTRACT IN KOREAN 79
Table 2.1. Biophysical characterization of various concentration of fucoidan. 47
Figure 1.1. Schematic illustration of the experimental procedure for the... 21
Figure 1.2. Characterization of NVs and FT/NVs. (A) Total amount of... 22
Figure 1.3. NVs and FT/NVs suppressed TNF-α-induced inflammation in... 24
Figure 1.4. Nanovesicles reduced the production of pro-inflammatory... 26
Figure 1.5. Nanovesicles inhibited the TNF-α-induced oxidative stress.... 29
Figure 1.6. NVs and FT/NVs diminished TNF-α-induced NF-κB... 31
Figure 2.1. Characterization of accumulated ECM after treated with... 51
Figure 2.2. Identification of ECM deposition after decellularization. (A)... 55
Figure 2.3. Characterization of pre-osteoblast derived EVs. (A) Size... 57
Figure 2.4. Effect of decellularized ECM and EVs for osteogenic... 60