Title Page

ABSTRACT

Contents

List of Abbreviations 16

Chapter 1. General Introduction 20

1.1. Advanced Polymeric Hydrogels for Tissue Engineering and Regenerative Medicine 20

1.1.1. Trends in Tissue Engineering and Regenerative Medicine 20

1.1.2. Bioactive Polymeric Hydrogels for In Situ Tissue Regeneration 21

1.2. Understanding the Endogenous Wound Healing Process and Foreign Body Reaction 23

1.2.1. Innate Wound Healing Process 23

1.2.2. Foreign Body Reaction 27

1.3. Oxygen in Wound Healing 29

1.4. Hyperoxic Oxygen and Its Biological Mechanism 32

1.5. Recent Trends in Developing Oxygen-delivering Hydrogels 35

1.5.1. Oxygen-releasing Hydrogels 36

1.5.2. Oxygen-generating Hydrogels 44

1.5.3. Strategies of Oxygen-delivering Hydrogels for Wound Healing 55

1.6. Current Limitations and Requirements for Advances in Developing Oxygen-delivering Hydrogels 63

1.6.1. Cytotoxicity and Complexity of Current Techniques 63

1.6.2. Therapeutic Strategies of Recent ODGs for Tissue Regeneration 63

1.7. Overall Objectives 64

Chapter 2. Self Cross-linkable Oxygen-generating Biosealant to Immobilize Stem Cell Spheroid-laden 3D Patch for Myocardial Infarction Treatment 66

2.1. Introduction 66

2.2. Experimental Section 70

2.2.1. Materials 70

2.2.2. Synthesis and Characterization of Biosealant Polymers 70

2.2.3. Fabrication of Tissue Adhesive Biosealant 72

2.2.4. Rheological Analysis of Biosealant 72

2.2.5. Tissue Adhesive Test of Biosealant 73

2.2.6. Cytocompatibility of Biosealant 73

2.2.7. In Vivo Biodegradability and Tissue Compatibility of Biosealant 74

2.2.8. Fabrication of S_3DP 75

2.2.9. Characterization of Spheroid Morphology 75

2.2.10. Quantitative Reverse Transcription-polymerase Chain Reaction 77

2.2.11. Protein Analysis using a Western Blot Analysis 77

2.2.12. Angiogenesis Analysis 79

2.2.13. Acute Rat MI Models and Transplantation of S_3DP In Vivo 79

2.2.14. Echocardiographic Evaluation 81

2.2.15. Histological Analysis 81

2.2.16. Statistical Analysis 82

2.3. Results and Discussion 83

2.3.1. Fabrication and Characterization of Tissue Adhesive Biosealant 83

2.3.2. Controllable Oxygen Generation of Biosealant 87

2.3.3. Biocompatible and Biodegradable Biosealant 89

2.3.4. Fabrication of S_3DP with Dual Pockets 91

2.3.5. Angiogenic Paracrine Effect of S_3DP 94

2.3.6. Therapeutic Effects of S_3DP on Cardiac Infarction 96

2.3.7. Reduced Cardiac Fibrosis of S_3DP on Cardiac Infarction 98

2.4. Conclusion 101

Chapter 3. Catalase-immobilized Oxygen-supplying Syringe to Fabricate Hyperoxia-inducible Hydrogels for In Situ Tissue Regeneration 102

3.1. Introduction 102

3.2. Experimental Section 106

3.2.1. Materials 106

3.2.2. Oxyringe Fabrication 106

3.2.3. Characterization of the PDA-modified syringe 107

3.2.4. Catalase Activity of Oxyringe 107

3.2.5. Oxygen-generating Kinetics of Oxyringe 111

3.2.6. Synthesis and Characterization of GtnSH and GelMA 111

3.2.7. Hydrogel Fabrication and Its Phase Transition 112

3.2.8. Oxygen-releasing Kinetics of Hydrogels In Vivo 113

3.2.9. Rheological Analysis of Hydrogels 115

3.2.10. H₂O₂ Measurements of Oxyringe and Oxygen-releasing Hydrogel 115

3.2.11. Cytocompatibility Test of Hyperoxia-inducible Hydrogels 116

3.2.12. In Vivo Hemostatic Ability Test 116

3.2.13. Subcutaneous Implantation In Vivo 117

3.2.14. In Vivo Wound Remodeling Evaluation 117

3.2.15. Histological Analysis 118

3.2.16. Statistical Analysis 118

3.3. Results and Discussion 119

3.3.1. PDA-Conjugated Syringe to Immobilize Catalase in an Oxyringe 119

3.3.2. Oxyringe Fabrication and Its Controllable Oxygen Generation 122

3.3.3. Fabrication and Characterizations of Oxygen-releasing Hydrogels 125

3.3.4. Cytocompatible Oxygen-releasing Matrices by H₂O₂ Scavenging 129

3.3.5. Hyperoxia-inducible Hydrogel as a Hemostatic Physical Barrier 133

3.3.6. Boosted Inflammation and Proliferation Phases with Hyperoxia-inducible Hydrogels 135

3.3.7. Promoted Wound Remodeling Phase via Hyperoxic Condition 139

3.4. Conclusion 141

Chapter 4. Applicability of Oxygen-supplying Technique for Fabricating Various Oxygen-delivering Platforms 142

4.1. Introduction 142

4.2. Experimental Section 144

4.2.1. Materials 144

4.2.2. Oxygener Fabrication 145

4.2.3. Characterization of the PDA-coated Container 145

4.2.4. H₂O₂-scavenging Ability of Oxygener 145

4.2.5. Synthesis and Characterization of GtnSH and GtnMI 147

4.2.6. Oxygen-generating and -supplying Capacity of Oxygener In Vitro 148

4.2.7. Fabrication of Cytocompatible Oxygen-enriched Media 148

4.2.8. In Vivo Wound Healing Evaluation and Biocompatibility Test 149

4.2.9. Total mRNA Sequencing 150

4.2.10. Histological Analysis 150

4.2.11. Statistical analysis 151

4.3. Results and Discussion 152

4.3.1. Fabrication of Oxygener via Improved PDA Coating Efficiency 152

4.3.2. Promoted H₂O₂ Scavenging and Oxygen Generation of Oxygener 155

4.3.3. Cytocompatible Oxygen-enriched Media as Hyperoxic Cell Culture Conditions 158

4.3.4. Fabrication of Hyperoxic Oxygen-releasing Hydrogels 160

4.3.5. Facilitated Wound Healing via Hyperoxic Oxidative Stress 162

4.3.6. Rapid Transition of Inflammation Phase into Proliferation Phase 164

4.3.7. Expedited Wound Remodeling Phase 167

4.4. Conclusion 170

Chapter 5. Significance and Future Perspective 171

5.1. Novelty of Our Systems 171

5.2. Future Direction 172

References 174

국문초록 209

Table 1.1. Summary of oxygen tension in various tissues and physiological conditions 34

Table 1.2. Characterization and application of various ODGs 56

Table 1.3. Summary of recent ODGs for TERM 58

Table 2.1. Sample codes and experimental condition of S_3DP. 76

Table 2.2. Sequences for primers used in qRT-PCR. 78

Table 2.3. Sample codes and compositions in animal study. 80

Table 3.1. Condition of catalase immobilization in Oxyringe. 108

Table 3.2. Sample codes and compositions of PDA-modified syringe (Final concentrations). 110

Table 3.3. Composition of hyperoxia-inducible hydrogels (final concentrations). 114

Table 4.1. Condition of catalase immobilization in Oxygener. 146

Fig. 1.1. Advanced polymeric hydrogels for in situ tissue regeneration. (a) Trends in TERM. (b) Bioactive polymeric hydrogels for in situ tissue regeneration. 22

Fig. 1.2. Stages of innate wound healing processes. (a) Blood clot formation as a biological barrier. (b) Immune cell recruitment and elimination of debris and foreign bodies. (c) Fibroblast-... 26

Fig. 1.3. Foreign body reaction to biomaterials. (a) Non-specific protein adsorption of blood- derived proteins, such as complement and fibronectin, on biomaterial surfaces. (b) Chemotactic... 28

Fig. 1.4. The roles of oxygen in tissue regeneration. (a) Improving cell viability and metabolic activity under hypoxic conditions. (b) Enhanced immune cell mobilization and its inflammatory... 31

Fig. 1.5. Molecular mechanism of hyperoxia in tissue regeneration 33

Fig. 1.6. Oxygen-delivering hydrogels for in situ tissue regeneration. (a) Oxygen-releasing hydrogels incorporating various oxygen carriers. (b) Oxygen-generating hydrogels utilizing... 37

Fig. 1.7. HBOCs as engineered natural oxygen carriers. (a) Layer-by-layer technique for preserving stability and oxygen release of Hbs. (b) Schematic illustration of the photothermal Hb-... 40

Fig. 1.8. PFCs as artificial oxygen carriers (a) Scheme of oxygen-enriched nanoerythrocytes fabrication. (b) Increase of oxygen tension after treatment of nanoerythrocytes. (c) Hypoxia... 43

Fig. 1.9. Solid peroxide as an oxygen-producing crosslinker. (a) Design of OGA hydrogel formation via CaO₂-mediated dual function. (b) Oxygen-generating behavior of OGA depending... 47

Fig. 1.10. The combination of liquid peroxide and catalase. (a) Schematic representation of oxygen-producing liposomes (H₂O₂ @Lip and CAT@Lip). (b) Oxygen-releasing profile of... 50

Fig. 1.11. Photosynthetic microalgae as an emerging oxygen generator. (a) Digital images of the microalgae-encapsulated scaffold with SEM analysis. (b) biocompatibility and proliferation of... 54

Fig. 1.12. Development of a new type of oxygen-supplying platform and hyperoxia-inducible hydrogels via CaO₂-mediated catalase immobilization. (a) Fabrication of self cross-linkable... 65

Fig. 2.1. Fabrication of S_3DP with dual pockets and their therapeutic outcomes. (a) The formation of hADSC spheroids within the open or combined open/closed pockets of the 3D patch... 69

Fig. 2.2. Synthesis and characterization of GtnSH and GtnMI. (a) Schematic illustration of GtnSH and GtnMI synthesis using EDC/NHS chemistry. ¹H NMR spectra of (b) GtnSH and (c) GtnMI... 84

Fig. 2.3. Fabrication and characterization of Gtn-based biosealant. (a) Schematic illustration of biosealant for fixing S_3DP. (b) Mechanism of hydrogel formation and tissue adhesion. (c)... 86

Fig. 2.4. Controllable oxygen generation of biosealant. (a) Schematic illustration of oxygen measurement. (b) Oxygen-generating kinetic with various CaO₂ concentrations. 88

Fig. 2.5. Biocompatible and biodegradable biosealant. Cytocompatibility of (a) polymer and (b) biosealant on HDFs. (c) Fluorescence images of the HDFs after a day of hydrogel eluates. (d)... 90

Fig. 2.6. Fabrication and characterization of S_3DP as cytocompatible 3D spheroid culture system. (a) Scheme of the hADSCs spheroid fabrication. (b) Representative optical images of hADSCs... 93

Fig. 2.7. Angiogenic paracrine effects of hADSC spheroids in the open pocket and open/closed pocket patches. (a) The relative gene expression of HIF-1α. The relative expression of (b) genes... 95

Fig. 2.8. Transplantation of S_3DP in rat MI model for improving cardiac functions. (a) Representative photographs of the heart after acute MI modeling and transplantation of S_3DP... 97

Fig. 2.9. Reduced cardiac fibrosis of the S_3DP in vivo. Schematic illustration showing (a) the location of areas 1 to 3 in the heart and (b) the cross-section of the heart. (c) Representative images... 100

Fig. 3.1. Oxyringe as a new type of oxygen-supplying system to create hyperoxia-inducible hydrogels. (a) Schematic representation of Oxyringe and (b) hyperoxia-inducible hydrogel... 105

Fig. 3.2. The procedure of Oxyringe fabrication. (a) PDA coating onto the inner surface of the syringe. (b) Catalase immobilization within the PDA-conjugated syringe. Reproduced with... 109

Fig. 3.3. PDA surface modification of syringe to fabricate Oxyringe. (a) Schematic representation of Oxyringe fabrication via CaO₂-mediated PDA surface modification and catalase immobilization.... 121

Fig. 3.4. Controllable oxygen generation of Oxyringe. Quantification of immobilized catalase activity depends on (a) immobilization temperature, (b) immobilization time, and (c) feed... 124

Fig. 3.5. Characterization of GtnSH and GelMA. ¹H NMR spectra of Gtn, GtnSH, and GelMA with a degree of substitution on the gelatin backbone. 126

Fig. 3.6. Synthesis and characterization of hyperoxia-inducible hydrogels. (a) Schematic illustration of oxygen-releasing hydrogel fabrication using an Oxyringe. b-d) Effect of H₂O₂... 128

Fig. 3.7. Cytocompatible hyperoxia-inducible hydrogels through residual H₂O₂ decomposition. (a) H₂O₂ -decomposing profile of Oxyringe with various concentrations of H₂O₂ and incubation... 131

Fig. 3.8. DO tension of NG group. 132

Fig. 3.9. Hemostatic physical barriers. (a) Scheme of hemostasis analysis. (b) Macroscopic images of liver bleeding model in control, NG, and HG groups. (c) Quantitative analysis of... 134

Fig. 3.10. Injectable hyperoxia-inducible hydrogels and their host cell infiltration. (a) Rheological analysis of hydrogels. (b) Process of subcutaneous injection and (c) images of mice with harvested... 136

Fig. 3.11. Accelerated initial macrophage recruitment and its rapid progression into the proliferation phase. (a) Scheme of subcutaneous hydrogel implantation and in vivo oxygen... 138

Fig. 3.12. Improved fibroblast differentiation and structural functionality of regenerated skin tissue. (a) Schematic illustration of wound remodeling tests using subcutaneous hydrogel... 140

Fig. 4.1. Fabrication of Oxygener via improved CaO₂-mediated DA oxidation. (a) Schematic representation of Oxygener fabrication via CaO₂-mediated PDA coating and catalase... 154

Fig. 4.2. Oxygener as an improved H₂O₂-scavenging and controllable oxygen-generating system. (a) Schematic illustration of catalase immobilization in Oxygener. H₂O₂ -scavenging kinetics of... 157

Fig. 4.3. Cytocompatible oxygen-enriched media as hyperoxic conditions for 3D cell culture condition in vitro. (a) Schematic illustration of oxygen measurement with the 3D cell culture... 159

Fig. 4.4. Fabrication of hyperoxic oxygen-releasing hydrogels via immersion in oxygen-enriched media. (a) Schematic illustration of oxygen measurement within hydrogels. (b) Oxygen... 161

Fig. 4.5. Biocompatible hyperoxic oxygen-releasing platforms for efficient wound healing. (a) Schematic illustration of wound closing test and oxygen measurement using a mouse critical... 163

Fig. 4.6. Hyperoxic condition for accelerating phase transition from inflammation to proliferation. Fluorescence images of explanted hydrogels with surrounding tissue after 7 days were stained... 166

Fig. 4.7. Facilitated fibroblast differentiation and collagen maturation in wound remodeling. Harvested skin tissues after 2 weeks were stained with (a) α-SMA for fibroblast differentiation... 169