Cryo-GelMA 是一類水凝膠，最近在藥物傳輸和細胞治療領域引起了人們的廣泛興趣，因為它具有高度互連的多孔結構和高含水量，提供與組織的物理相似性和良好的封裝親水性藥物的能力。 在第一項工作中，Cryo-GelMA 應用於聯合化療（阿黴素）、免疫療法（負載 AuND-LPS 的骨髓源性樹突狀細胞 (BMDC) 和抗 PD1 阻斷）以預防術後腫瘤復發。 該研究最初著重於以金奈米點（AuND-LPS）修飾脂多醣（LPS）以增強其免疫刺激特性。 這種修飾對骨髓源性樹突細胞 (BMDC) 的 Th1 極化產生正面影響，同時減少 Th2 極化。 AuND-LPS 也透過上調 CCR7 受體來改善 BMDC 歸巢至引流淋巴結。 阿黴素與 AuND-LPS 組合顯示出協同效應，上調 cGAS-STING 路徑並增強樹突狀細胞成熟。 此後，在體內原位乳腺腫瘤模型中，生物材料疫苗結節將阿黴素和負載 AuND-LPS 的 BMDC 封裝在 Cryo-GelMA 基質中，有效減緩了原發腫瘤的生長並抑制了遠端轉移。 基於樹突狀細胞的疫苗增加了發炎細胞因子的表達，活化了抗腫瘤免疫細胞，並與抗PD1阻斷療法相容，協同增強了針對腫瘤生長的免疫力。
在第二項研究中，探索了一種在不改變其固有特性的情況下增強 Cryo-GelMA 機械強度的方法。 將 Cryo-GelMA 整合到 3D 列印的陀螺儀結構中，形成了混合支架。 混合支架保留了傳統 Cryo-GelMA 的有利特性，例如高孔隙率、互連性和形狀恢復，表現出剛度的協同增強。 混合支架的壓縮模量超過了傳統 Cryo-GelMA 或其單獨框架。 這種新穎的方法提供了一種提高 Cryo-GelMA 類材料機械強度的方法。 華頓商學院的果凍間質幹細胞 (WJMSC) 表現出與混合支架的相容性，表現出高細胞附著、均勻分佈和增殖。 螺旋體結構利用混合支架內這種結構固有的高滲透性，促進細胞滲透。

Cryo-GelMA, a class of hydrogel, has recently attracted much interest in drug delivery and cell therapy as it possesses a highly interconnected porous structure with high water content providing physical similarity to tissues and good capability to encapsulate hydrophilic drugs. In the first work, the Cryo-GelMA was applied in co-delivering chemotherapy (Doxorubicin), immunotherapy (AuND-LPS-loaded bone-marrow-derived dendritic cells (BMDCs) and anti-PD1 blockade) for preventing post-surgery tumor recurrence. The study initially focused on modifying Lipopolysaccharide (LPS) with Au nanodots (AuND-LPS) to enhance its immunostimulatory properties. This modification positively influenced the Th1 polarization of bone marrow-derived dendritic cells (BMDCs) while reducing Th2 polarization. AuND-LPS also improved BMDC homing to draining lymph nodes by upregulating the CCR7 receptor. Combining doxorubicin with AuND-LPS demonstrated a synergistic effect, upregulating the cGAS-STING pathway and enhancing dendritic cell maturation. After that, in an in vivo orthotopic breast tumor model, biomaterial vaccine nodules, encapsulating doxorubicin and AuND-LPS-loaded BMDCs in a Cryo-GelMA matrix, effectively slowed primary tumor growth and suppressed distal metastasis. The dendritic cell-based vaccine increased inflammatory cytokine expression, activated anti-tumor immune cells, and showed compatibility with anti-PD1 blockade therapy, synergistically enhancing immunity against tumor growth.
In the second study, a methodology was explored to enhance the mechanical strength of Cryo-GelMA without altering its intrinsic properties. The integration of Cryo-GelMA into a 3D printed gyroid structure resulted in the formation of hybrid scaffolds. Preserving the advantageous features of conventional Cryo-GelMAs, such as high porosity, interconnectivity, and shape recovery, the hybrid scaffolds exhibited synergistic enhancement in stiffness. The compressive modulus of the hybrid scaffolds surpassed that of conventional Cryo-GelMA or its individual frameworks. This novel approach offers a means to improve the mechanical strength of Cryo-GelMA-like materials. Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) exhibited compatibility with the hybrid scaffolds, demonstrating high cell attachment, even distribution, and proliferation. The gyroid architecture facilitated cell penetration, leveraging the high permeability inherent in this structure within the hybrid scaffolds.

Table of Contents
Abstract 3
Table of Contents 5
List of Tables 10
List of Figures 11
List of Supplementary Figure 17
List of acronyms 18
1. Introduction and Research Background 19
1.1 Cryo-GelMA versus Conventional Hydrogel 19
1.2 Application of Cryo-GelMA in Drug Delivery System 21
1.3 Application of Cryogels in Tissue Regeneration and Its Limitations 22
1.4 Motivation 25
2. GelMA Cryogel as a Delivery System for Co-delivering Chemotherapy and Dendritic Cell-based Therapy for Recurrent Breast Tumors 27
2.1 Introduction to Breast Cancer and Dendritic Cell-based Therapy 27
2.1.1 Breast Cancer and Limitations of Current Treatments 27
2.1.2 Dendritic Cells and Biomaterial Scaffold-Assisted Dendritic Cell-based Immunotherapy 30
2.1.3 Lippolysaccharide as an Immune Adjuvant 32
2.1.4 Combination of Chemotherapy and Immunotherapy for Cancer Treatment 35
2.2 Experiment Design and Methods 36
2.2.1 Cells and Material 36
2.2.2 Synthesis of GelMA and GelMA Cryogelation 37
2.2.3 Bone-Marrow-derived Dendritic Cell Culture and Treatment. 38
2.2.4 Preparation of AuNDs 38
2.2.5 In-vitro Release Profile of Doxorubicin from GelMA Cryogel: 39
2.2.6 Transmission Electron Microscopy: 39
2.2.7 Animals and Tumor Models: 39
2.2.8 Flow Cytometry: 39
2.2.9 Real-Time PCR: 40
2.2.10 IL-12 and IL-10 Enzyme-Linked Immunosorbent Assay. 41
2.2.11 In Vitro Study of STING Activation and DC maturation induced by Cancer Cell Medium and AuND-LPS 41
2.2.12 Transwell Assay for BMDCs migration. 42
2.3 Result and Discussion 42
2.3.1 Modulation of Cytokine and Chemokine Expression in Dendritic Cell by AuND-LPS 42
2.3.2 Delivery of BMDCs and AuND-LPS@DCs by Cryo-GelMA into Mice 46
2.3.3 Combination of Doxorubicin and AuND-LPS Up Regulate The cGAS-STING Pathway 49
2.3.4 Cryo-GelMA-derived Vaccine Inhibiting Tumor Recurrence In Vivo 52
2.3.5 Modulating The Cytokines Level and Immune Cells of Post-surgery Tumor and Lymph Node by Cryo-GelMA loading Doxorubicin and AuND-LPS-treated BMDCs 54
2.3.6 Dox+AuND-LPS@DCs Synergy with Immune Checkpoint PD-1 Therapy to treat breast tumor post-surgery 58
3. Reinforcement of Cryo-GelMA by 3D-printing framework. 61
3.1 Introduction to Application of Cryo-GelMAs and 3D Printing in Tissue Regeneration 61
3.1.1 Biomaterial Scaffold-Assisted Tissue Engineering: 61
3.1.2 Introduction to DLP-AM 63
3.2 Experiment Design and Methods 65
3.2.1 Synthesis of POLYCAPROLACTONE DIACRYLATE 65
3.2.2 Synthesis of POLYETHYLENE DIACRYLATE 66
3.2.3 Chemical Characterization of GelMA and POLYCAPROLACTONE DIACRYLATE: 66
3.2.4 Design of Gyroid Scaffolds: 67
3.2.5 Fabrication of Three Dimensional-Printed Frameworks: 67
3.2.6 Fabricating Hybrid Composite Scaffold 68
3.2.7 Porosity 68
3.2.8 Measurement of Recovery Ability 69
3.2.9 Compression Test: 70
3.2.10 Cell Culture on Scaffolds 70
3.2.11 Preparation of Sample for Scanning Electron Microscope (SEM): 71
3.2.12 Resazurin Reduction Assay 71
3.2.13 Determination of Theoretical Permeability 72
3.2.14 Statistical Analysis 73
3.3 Result and Discussion 75
3.3.1 Creating and Characterizing the Printed Gyroid Structure 75
3.3.2 Characterization of Composite Scaffold 77
3.3.3 Physical Properties of Hybrid Scaffolds 80
3.3.4 Mechanical Property of 3D-Printed and Hybrid Scaffold: 81
3.3.5 Controlling the Mechanical Strength of Composite Scaffold Through the Structure of Three-Dimensional Framework. 83
3.3.6 WJMSC Proliferation on Hybrid Scaffold and Cryo-GelMA 87
3.3.7 WJMSC Penetration in Hybrid Scaffold and Cryo-GelMA 90
4. Conclusion 93
5. Future Works 95
6. Referrences 97
7. Supplementary Figure 107

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