마이크로 구조를 갖는 조직 구조체 제작을 위한 Cascade Bioprinting 기술 개발 = Development of Cascade Bioprinting Technique for Microstructured Tissue Fabrication

다국어 초록 (Multilingual Abstract)

The number of patients waiting for organ transplants continues to rise; however, the scarcity of donor organs leads to an increasing number of deaths among patients awaiting transplantation each year. Furthermore, animal testing, which is performed to evaluate the safety and efficacy of drugs, cosmetics, and chemicals for human use, is continuously increasing, raising significant ethical concerns. Therefore, there is a critical need for artificial tissues/organs capable of substituting damaged or dysfunctional human tissues and replacing animal experiments. Successful artificial tissues/organs must closely replicate the functionality of human tissues. Recently, the importance of mimicking microstructures present in native tissues has been emphasized for achieving functional artificial tissues, necessitating the development of technologies capable of replicating these intricate in vivo microstructures. Three-dimensional (3D) bioprinting technology, employing program-controlled 3D printing systems, allows precise positioning of cells to fabricate three-dimensional artificial tissues. Using this technology, layered structures resembling blood vessels and skin in the human body have been successfully recreated, resulting in highly functional artificial tissues. Nevertheless, conventional 3D bioprinting methods exhibit limited precision, preventing the accurate replication of heterogeneous microstructures such as liver lobules or capillary networks. Although multimaterial bioprinting techniques have been developed to address this limitation, existing challenges, such as syringe size constraints, low hydrogel stiffness, and specialized stacking processes, still impede the creation of complex microstructures similar to those found in vivo.
To address these challenges, this study developed a novel 3D bioprinting technology, referred to as “Cascade Bioprinting”. In this method, highly viscous hydrogels are extruded through small-diameter nozzles at low velocities, resulting in low Reynolds number flows characterized by dominant viscous forces. Under these conditions, two different fluids flowing simultaneously through a single channel do not mix significantly. Utilizing this phenomenon, hydrogels pre-patterned in syringes can maintain their spatial arrangements upon extrusion, resulting in scaled-down patterns without mixing. Repeating this process, by reprinting previously extruded patterns within syringes containing different hydrogels, further reduces pattern sizes while preserving structural integrity. Two strategies for pre-positioning hydrogels within syringes were proposed: direct printing within syringes using nozzles and transferring patterns from 3D-printed precursor cartridges into syringes. Experimental evaluations demonstrated how these methods influenced the formation of cross-sectional patterns in the reprinted biostructures. Cascade bioprinting showed high precision, capable of creating heterogeneous patterns under 50 µm by exploiting laminar flow characteristics. Additionally, the reprinting process generated extensional flow, promoting the alignment of biomaterials and facilitating directional cell alignment within printed filaments. The capability of this technique to replicate heterogeneous microstructures similar to spinal cord grey matter and muscle fiber bundles was demonstrated, and successful bioprinting of highly prevascularized hepatic tissues was achieved.
However, during the development of cascade bioprinting, inconsistencies were observed between syringe-initialized patterns and resultant filament cross-sections, requiring extensive trial and error regarding cartridge shape and hydrogel selection. To improve efficiency, a computational fluid dynamics (CFD) model was developed to predict bioprinting outcomes. This model, leveraging laminar flow characteristics inherent to cascade bioprinting, accurately predicted filament cross-sections and identified critical process parameters for optimized pattern formation, enabling effective pre-design of bioprinting processes.
The liver plays a crucial role in drug metabolism, making artificial liver development essential for organ replacement and drug screening applications. The liver's highly developed vascular network significantly enhances metabolic efficiency by facilitating effective material transport. However, traditional biofabrication methods face limitations in achieving required vascular dimensions due to low spatial resolution. While conventional multimaterial bioprinting techniques can produce vascular structures within printed tissues, they fall short in efficiently reproducing dense, complex vascular networks comparable to native tissues. The developed cascade bioprinting technology effectively overcomes these challenges, demonstrating high precision and simplicity in creating complex native tissue-like microstructures. Utilizing the developed CFD model for optimized process parameter design, a liver tissue construct featuring high-density microvascular structures was efficiently fabricated. The fabricated microstructured hepatic tissues exhibited significantly enhanced protein synthesis, drug and toxin metabolism, and angiogenic potential compared to homogeneous or simply vascularized tissues.
In conclusion, cascade bioprinting represents a highly effective multimaterial bioprinting method for fabricating intricately microstructured tissue constructs, showcasing substantial potential for application in artificial organs and drug screening models with superior functionality.

목차 (Table of Contents)

• List of Figures 1
• List of Tables 4
• Abstract 5
• 1. Introduction 8
• 1-1. Background 8
• 1-2. Research Trends. 13
• 1-3. Research Objective 18
• 1-4. Dissertation Organization 19
• 2. Development and Characterization of Cascade Bioprinting Technique 21
• 2-1. Introduction 21
• 2-2. Results and Discussion 24
• 2-2-1. Cascade bioprinting 24
• 2-2-2. Characterization for cartridge-free method 26
• 2-2-3. Characterization of cartridge-based method 29
• 2-2-4. Evaluation of advantages of cascade bioprinting 32
• 2-2-5. Bioprinted constructs via cascade bioprinting 36
• 2-3. Summary 41
• 3. Development of Computational Fluid Dynamics Model and Investigation of Process Parameters for Cascade Bioprinting 42
• 3-1. Introduction 42
• 3-2. Results and Discussion 45
• 3-2-1. Establishment of CFD model 45
• 3-2-2. Application of CFD model to reprinting process 48
• 3-2-3. Effect of pressure 50
• 3-2-4. Effect of nozzle diameter 52
• 3-2-5. Effect of nozzle shape 54
• 3-2-6. Effect of syringe outlet angle 57
• 3-2-7. Influence of height of precursor cartridge 59
• 3-2-8. Fabrication of structures with complex cross-sectional geometries 61
• 3-3. Summary 64
• 4. Fabrication of Highly Vascularized, Microstructured Hepatic Tissues using Cascade Bioprinting Technique 65
• 4-1. Introduction 65
• 4-2. Results and Discussion 70
• 4-2-1. Design of cascade bioprinting process 70
• 4-2-2. Fabrication of highly vascularized hepatic tissue 73
• 4-2-3. Assessment of hepatic tissue functionality 76
• 4-2-4. Evaluation of metabolic ability 78
• 4-2-5. Assessment of angiogenic ability 81
• 4-3. Summary 84
• 5. Conclusion and Future Works 85
• 5-1. Summary and Conclusion 85
• 5-2. Future Works 86
• Experimental Section 87
• References 98
• 한글 요약문 (Summary in Korean) 108