Title Page
ABSTRACT
Contents
List of Abbreviations 12
Chapter 1. General Introduction 15
1.1. Atopic Dermatitis 15
1.1.1. Pathogenesis and Microenvironments of Atopic Dermatitis 18
1.1.2. Chronic Hypoxia in Atopic Dermatitis 22
1.2. Engineered Pre-clinical Skin Models 24
1.2.1. Limitation of Current Skin Models 25
1.3. Polymeric Hydrogels 29
1.3.1. In Situ Cross-linkable Hydrogels as Artificial ECM Scaffolds 31
1.4. Research Motivation and Approaches 37
Chapter 2. Hydrogel-based Oxygen-controllable In Vitro Models 39
2.1. Introduction 39
2.2. Experimental Section 42
2.2.1. Materials 42
2.2.2. Synthesis and Characterization of GtnSH and GtnMI Polymer 43
2.2.3. Preparation of the GtnSH/GtnMI Hydrogels and Phase Transition Time 45
2.2.4. Rheological Analysis of the GtnSH/GtnMI Hydrogels 47
2.2.5. 3D Cell Encapsulation using GtnSH/GtnMI Hydrogels 47
2.2.6. Cytocompatibility of the GtnSH/GtnMI Hydrogels (Live/dead assay) 50
2.2.7. Real-time Oxygen Measurement Monitoring System 50
2.2.8. RNA Isolation and Gene Expression Analysis (qRT-PCR) 50
2.2.9. Immunofluorescence Staining of Periostin 54
2.2.10. Periostin Protein Expression Analysis 54
2.2.11. Drug Screening Platform using Model Drugs 55
2.2.12. RNA Sequencing Analysis (RNA-seq) 55
2.2.13. Statistical Analysis 56
2.3. Results and Discussion 57
2.3.1. Synthesis and Characterization of GtnSH and GtnMI 57
2.3.2. Fabrication and Characterization of GtnSH/GtnMI Hydrogels 60
2.3.3. Cytocompatible Hydrogel-based 3D Cell Constructs 64
2.3.4. Recapitulation of Hypoxic Environments 66
2.3.5. In Vitro Atopic Dermatitis Models 68
2.3.6. Drug Screening Platform using Model Drugs 71
2.3.7. RNA Sequencing Analysis (RNA-seq) 73
Chapter 3. Conclusion and Future Direction 79
Supplementary data 80
References 82
국문초록 95
Table 1.1. Preclinical skin models. 28
Table 1.2. Desirable properties of hydrogel-based scaffolds. 36
Table 2.1. Sample code and the hydrogels composition. 46
Table 2.2. 3D cell encapsulation conditions. 49
Table 2.3. The sequences of the primers used in the qRT-PCR analysis. 52
Table 2.4. Sample code and concentration used in the experiment. 53
Fig. 1.1. Global atopic dermatitis market with Ref. 17
Fig. 1.2. Stage-based pathogenesis and main mechanisms of atopic dermatitis. Reproduced with permission from [1]. Copyright (2018) Springer Nature. 20
Fig. 1.3. Various factors and cells affect itching in atopic dermatitis. Periostin, a representative factor involved in itching. Adapted with permission from [18]. Copyright (2019) BLACKWELL... 21
Fig. 1.4. Diagram demonstrating the various factors relevance of preclinical skin models. Adapted with permission from [54]. Copyright (2021) Multidisciplinary Digital Publishing Institute. 27
Fig. 1.5. Fabrication and applications of in situ cross-linkable hydrogels. Adapted with permission from [58]. Copyright (2021) Multidisciplinary Digital Publishing Institute. Adapted with... 34
Fig. 1.6. Considerations and properties of recapitulating native ECMs. Adapted with permission from [117]. Copyright (2022) Multidisciplinary Digital Publishing Institute. Reproduced with... 35
Fig. 1.7. The schematic representation of our approach to recapitulate the atopic dermatitis microenvironments via hydrogel-based engineered skin models. Schematic diagram of hydrogel-... 38
Fig. 2.1. Synthesis of GtnSH polymer. (a) Schematic representation of synthesis thiolated gelatin (GtnSH). (b) The chemical structure of gelatin and GtnSH through ¹H NMR analysis. (c) Ellman's... 58
Fig. 2.2. Synthesis of GtnMI polymer. (a) Schematic representation of synthesis maleimide-conjugated gelatin (GtnMI). (b) The chemical structure of gelatin and GtnMI through ¹H-NMR... 59
Fig. 2.3. Fabrication and characterization of the GtnSH/GtnMI hydrogels. (a) Schematic representation of gel formation. (b) Digital images of the sol-gel phase transition. (c) Phase... 63
Fig. 2.4. 3D cell encapsulation and in vitro cytocompatibility of the hydrogels. (a) Schematic representations of 3D encapsulation hydrogels and confocal images of 3D encapsulation... 65
Fig. 2.5. Real-time oxygen measurement monitoring system and HIF-1α expression analysis. (a) Schematic representations of real-time O₂ monitoring system. (b) Result of O₂ measurement for... 67
Fig. 2.6. Periostin expression analysis. (a) Schematic representations of periostin expression analysis. Gene expression at (b) 21% pO₂ condition and (c) 5% pO₂ condition (qRT-PCR). The... 70
Fig. 2.7. Drug toxicity test and drug response analysis. (a) Schematic representations of drug screening platform using model drugs. (b) Live/dead images of HDFs cultured in dexamethasone-... 72
Fig. 2.8. RNA sequencing analysis. (a) PCA between hypoxia group (5%_25) and normoxia group (21%_25). (b) Volcano plots showing DEGs analysis in 5%_25 group compared to 21%_25 group... 75
Fig. 2.8. RNA sequencing analysis. ORA results analyzed using DEGs. (c) GO BP terms. (d) KEGG pathways. 76
Fig. 2.8. RNA sequencing analysis. (e) GSEA using GO BP terms and KEGG pathways. 77
Fig. 2.8. RNA sequencing analysis. (f) Marker gene analysis results and (g) volcano plot of AD-specific fibroblasts expressed under hypoxia group (5%_25) (padj ＜0.01; |log2FoldChange|＞ 0). 78
Fig. S1. Characterization of hydrogels. Phase transition time of hydrogel depending on (a) gelatin concentration, (b) the ratio of the polymer concentration, and (c) the ratio of the equivalent... 80
Fig. S2. Gene expression analysis (qRT-PCR). Tenascin-C expression at (a) 21% pO₂ condition and (b) 5% pO₂ condition. WNT5A expression at (c) 21% pO₂ condition and (d) 5% pO₂ condition.... 81