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About this book

This book presents a comprehensive study on microextrusion-based 3D bioprinting technologies for bioinks with various crosslinking mechanisms, chiefly focusing on the bioprinting process and bioink properties to provide readers with a better understanding of this state-of-the-art technology. Further, it summarizes a number of general criteria and research routes for microextrusion-based 3D bioprinting using three experimental studies based on shear-thinning, thermo-sensitive and non-viscous hydrogel bioinks. The book also presents sample applications in the areas of stem cells and cell matrix interaction.

The book highlights pioneering results in the development of bioprinting technologies and bioinks, which were published in high-quality journals such as Advanced Materials, Biofabrication and ACS Biomaterials Science & Engineering. These include an in-situ crosslinking strategy that overcomes the viscosity limits for bioinks, which is virtually impossible using conventional strategies, and can be generalized for other bioink formulations.

Table of Contents

Frontmatter

Chapter 1. Introduction

Abstract
The applications of 3D printing (3DP) in biomedicine vary with the ink materials and thus the final products. For example, prototyping fabricated from non-biocompatible plastic could be used in surgery planning or guidance. Another example is the 3D printed implants made of biocompatible but nondegradable materials, such as titanium. One more step further, researchers have applied degradable biomaterials to print implantable scaffolds that would guide the native tissue regeneration and thus achieve the ideal repair. What this thesis presents belongs to another stage, where cells and other biological elements are directly incorporated in the 3DP process to create a living product, which is termed as 3D bioprinting. This technology is supposed to lead to organ printing in the future, which might sound crazy but is actually happening. 
Liliang Ouyang

Chapter 2. 3D Bioprinting and Bioink: Background

Abstract
This chapter generally introduces the field of 3D bioprinting, starting with the related concepts and their history. The definitions of bioprinting and bioink are clarified based on the most recent literature. The brief history of this field is reviewed, and the technology trends are presented from different angles, which all indicate a booming development. Then a comprehensive overview of state of the art is presented in terms of bioprinting technologies, bioinks, and application areas. Given the inconsistent classification method in literature, here we classify the bioprinting techniques according to the dimensions of the building blocks, which meet the essential characteristics of this bottom-to-up methodology. This chapter ends with the general challenges and some perspectives from the author.
Liliang Ouyang

Chapter 3. Materials and Methods

Abstract
The goal of this chapter is to better understand the bioprinting process by theoretically analyzing the correlated topics and questions. It summarizes the general criteria and research route for bioprinting. Despite numerous microextrusion bioprinting strategies developed, very little knowledge has been defined or discovered in terms of the nature of bioprinting. We believe that these strategies share some common features (e.g., one-dimensional filament as building block) that would lead to a better understanding and exploitation of this technology. This chapter will first analyze the general process of microextrusion bioprinting step by step and extract critical questions from each step. The general criteria will be subsequently concluded in terms of structural fidelity and cell protection. Based on the bioink crosslinking mechanisms, such criteria will be used to guide the design of the bioprinting process, covering the filament formation, deposition, and structure stabilization. Overall, a research route will be presented, which will be used in the subsequent case studies. Moreover, this chapter will introduce some general methods used in the study regarding rheology, 3D printability, shear stress determination, and cellular characterization.
Liliang Ouyang

Chapter 4. 3D Bioprinting of Shear-Thinning Self-assembly Bioink

Abstract
When applying a new material as a bioink in microextrusion bioprinting, numerous considerations need to be addressed. As stated in Chap. 3, one of the primary considerations is printability, which normally encompasses the material’s ability to (i) be injected from a printhead (ii) undergo rapid gelation upon deposition, and (iii) exhibit suitable mechanical properties that would support the printed structure. Given this, we seek to explore hydrogel formulation that could meet these criteria. Specifically, in this chapter, we will apply a shear-thinning and rapidly self-healing guest–host hydrogel based on hyaluronic acid (HA) to microextrusion 3DP. Adamantane (Ad, guest) and β-cyclodextrin (CD, host) moieties are separately coupled to HA, to create two hydrogel precursors that form a supramolecular assembly upon mixing [1]. It is hypothesized that such formulation would allow for smooth extrusion and temporary stabilization post-extrusion because of the shear-thinning and self-healing properties, respectively. To enhance the structural integrity of the supramolecularly crosslinked hydrogel, we also introduce photo-crosslinkable groups onto the macromers. We then investigate how each type of crosslinking (guest–host, photo-crosslinking, and their combination) affects the printability of multilayer scaffolds. Post-crosslinking methods are also explored concerning structural integrity and stability over time. Printed structures can be further functionalized to support cell culture. Similar dual-crosslinking mechanisms based on supramolecular and covalent bonding may enable the development of 3D printable hydrogel bioinks from materials that cannot otherwise be printed.
Liliang Ouyang

Chapter 5. 3D Bioprinting of Thermal-Sensitive Bioink

Abstract
In this chapter, we aim to explore the property of a common thermal-sensitive bioink and its effects on structure printability and embryonic stem cells (ESCs) viability. Despite progress in bioinks development, the effect of bioink properties on the formation of 3D construct and cell damage during the extrusion process are poorly characterized. Moreover, the parameter optimization based on specific cell type might not be applicable to other types of cells, especially those with high sensibilities, such as ESCs. In this study, we systematically study the construct printability and cell viability in a temperature-controlled bioprinting process by using gelatin-alginate hybrid materials. A novel method is established to determine suitable conditions that could achieve both good printability and high cell viability. The rheological properties of the bioinks are evaluated to determine the gelation properties under different gelatin concentrations, testing temperatures and time. The printability of a lattice construct is characterized by using a semi-quantified method. The LIVE/DEADTM assay show that ESCs viability increased with the increase of printing temperature increased and decrease of gelatin concentration. Furthermore, a fitting exponential relationship was obtained between cell viability and induced shear stress. By defining the proper printability and acceptable viability range, a conjunction parameters region is obtained to guide the parameter choosing. This study will provide insight into the fine-tuning of 3D bioprinting process regarding the integrity of printed construct and incorporated cells, especially for easily damaged cells like ESCs.
Liliang Ouyang

Chapter 6. 3D Bioprinting of Non-viscous Bioink

Abstract
Photo-crosslinkable hydrogels have great potential as bioinks. These materials have been developed over the past few decades to encompass a wide range of properties, and they have been of significant interest for their applicability in cell encapsulation and tissue formation. Despite the plethora of photo-crosslinkable hydrogels under development in the biomaterials field, their application to bioprinting is hindered through their generally low initial viscosity and challenges in polymerizing fast enough to maintain printed structures. To overcome this limitation, photo-crosslinkable hydrogels have been combined with polymers that gel through other mechanisms, such as with supramolecular assembly, temperature, or exposure to ions. This is not ideal, as it alters the material environment for cells. To address these challenges in printing photo-crosslinkable materials, here we present a generalizable bioprinting method to enable 3D printing of hydrogel structures from photosensitive precursors. In this approach, we introduce the light through a photo-permeable capillary (e.g., silicon tubing, glass) to crosslink the hydrogel immediately prior to leaving the needle and before deposition, which we termed in situ crosslinking. Advantages to this approach are (i) that it does not include any viscosity modulation or copolymerization with other polymers, (ii) that it can be generalized to different photo-crosslinkable hydrogel formulations, (iii) that it permits the encapsulation of viable cells, and (iv) that it can be used to print heterogeneous and complex structures.
Liliang Ouyang

Chapter 7. Biological Characterization and Applications

Abstract
Chapters 46 introduce three bioprinting works using bioinks with different crosslinking mechanisms, mainly from the angles of structural printability and cell viability post-printing. This chapter will present further biological characterization and application based on the specific techniques studied before. Specifically, the printed construct using supramolecular bioinks in Chap. 4 exhibits excellent structure fidelity and mechanical properties and allows for cell adhesion, all of which indicate a promising tissue engineering scaffold. The work in Chap. 5 leads to a perfect balance between structural printability and cell viability by using the easy-accessed and biocompatible bioink, gelatin–alginate hybrid formulation. This technique will be used to further investigate the signal pathway activation and embryonic stem cells’ behavior in 3D-bioprinted constructs. The technology developed in Chap. 6 highlights the use of non-viscous bioinks and flexibility in formulation types and building block complexity. Given this, this work will be used to explore the effects of different ink types and other materials cues on cell behavior, such as morphology.
Liliang Ouyang

Chapter 8. Conclusions and Future Work

Abstract
This chapter presents a conclusion to the work described in this thesis. The main contributions and findings are summarized, followed by some suggestions regarding future directions in the field of bioprinting.
Liliang Ouyang
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