Yong He obtained his PhD degree in mechanical engineering at the ZheJiang University in 2008. He is currently a professor at College of Mechanical Engineering, ZheJiang University, China. He is also the deputy director of Key Lab of 3D Printing Process and Equipment of ZheJiang Province. His research is focused on the biofabrication with 3D printing especially on the building organs on chips. He has published more than 100 international journal papers and authorized over 30 patents. He has developed many special 3D printers for the fabrication of microfluidic devices, such as 3D sugar printer and 3D softmatter printer.
Qing Gao obtained his BSc in mechanical design, manufacturing and automation at Hefei University of Technology in 2012. In 2017 he obtained his PhD degree in mechanical manufacturing and automation at the ZheJiang University and continue working in the university as a postdoc. He engages in research on biomanufacturing, biological 3D printing, organ chips, etc. As a core member, he has developed a portable biological 3D printer and high-performance GelMA bio-ink and is committed to building a "material + equipment + service" integrated intelligent manufacturing product system.
Yifei Jin received his Ph.D. in mechanical engineering from the University of Florida in 2018, He joined the Department of Mechanical Engineering at the University of Nevada, ¿Reno as assistant professor in July 2019. His primary research interests mainly involve 3D bioprinting of living tissue constructs, 3D printing of hydrophobic functional materials, yield-stress fluids for 3D printing applications, stimuli-responsive materials for 4D printing applications, and fabrication of multi-layered capsules. His research emphasizes the coupling of materials and fabrication approaches to develop novel 3D printing techniques and understand the underlying physics during printing.
Preface xv
1 3D Bioprinting, A Powerful Tool for 3D Cells Assembling1
1.1 What Is 3D Bioprinting? 1
1.2 Evolution of 3D Bioprinting 3
1.3 Brief Classification of 3D Bioprinting 4
1.4 Evaluation of Bioinks 5
1.5 Outlook and Discussion 6
References 8
2 Representative 3D Bioprinting Approaches11
2.1 Introduction11
2.2 Inkjet Bioprinting 13
2.2.1 Mechanisms of Droplet Formation 14
2.2.1.1 Continuous-Inkjet Bioprinting 14
2.2.1.2 Drop-on-Demand Inkjet Bioprinting 15
2.2.1.3 Electrohydrodynamic Jet Bioprinting 16
2.2.2 Hydrogel-Based Bioinks for Inkjet Bioprinting 17
2.2.2.1 Material Properties for Inkjet Bioprinting Applications 18
2.2.2.2 Commonly Used Hydrogels in Inkjet Bioprinting 19
2.2.3 Representative Cell Printing Applications 20
2.2.3.1 Bone and Cartilage Tissues 21
2.2.3.2 Organoids 22
2.2.3.3 Skin Tissues 22
2.2.3.4 Vascular Networks 22
2.2.4 Summary 22
2.3 Extrusion Bioprinting 23
2.3.1 Mechanisms of Extruding Biocompatible Materials 23
2.3.2 Primary Extrusion Bioprinting Strategies 24
2.3.3 Main Categories of Extrudable Biomaterials 25
2.3.3.1 Hydrogels 25
2.3.3.2 Micro-Carriers 26
2.3.3.3 Cell Aggregates 27
2.3.3.4 Decellularized Matrix Components 28
2.3.4 Summary 28
2.4 Light-Based Bioprinting 28
2.4.1 Laser-Assisted Bioprinting 28
2.4.1.1 Mechanism 28
2.4.1.2 Materials 30
2.4.1.3 Biomedical Applications 30
2.4.2 Stereolithography 32
2.4.2.1 Mechanism 32
2.4.2.2 Materials 33
2.4.2.3 Biomedical Applications 33
2.4.3 Multi-Photon Polymerization 34
2.4.3.1 Mechanism 34
2.4.3.2 Materials 35
2.4.3.3 Biomedical Applications 35
2.4.4 Digital Light Projection 3D Printing 35
2.4.4.1 Mechanism 36
2.4.4.2 Materials 37
2.4.4.3 Biomedical Applications 37
2.4.5 Computed Axial Lithography 37
2.4.5.1 Mechanism 37
2.4.5.2 Materials and Biomedical Applications 37
2.4.6 Summary 38
References 38
3 Bioink Design: From Shape to Function47
3.1 Significance of Bioink Design 47
3.2 Categories of Bioink 47
3.3 Three Evaluation Criteria of Bioink 48
3.3.1 Printability 48
3.3.2 Mechanical Properties 48
3.3.3 Biocompatibility 48
3.4 Strategies for Enabling the Printability 49
3.4.1 Optimization of Cross-linking Sequence 49
3.4.2 Support Material-Assisted Bioprinting 50
3.4.3 Microgel-Based Bioink 50
3.5 Strategies for Bioink Reinforcement 50
3.5.1 Composite Bioink Design 50
3.5.2 Microfiber-Assisted Reinforcement 51
3.6 Strategies for Improving the Biocompatibility 51
3.7 Representative Bioink Design Case: GelMA-Based Bioinks 52
3.7.1 Property Characterization of the GelMA Bioink 52
3.7.2 3D Bioprinting of GelMA Bioinks with Dual Cross-linking Strategy 53
3.7.3 3D Bioprinting of GelMA Bioinks with Nanoclay as Support 55
3.8 Commercial Bioink 57
3.8.1 GelMA (EFL-GM Series) 58
3.8.2 Fluorescent GelMA (EFL-GM-F Series) 58
3.8.3 Porous GelMA (EFL-GM-PR Series) 60
3.8.4 HAMA (EFL-HAMA Series) 64
3.8.5 SilMA (EFL-SilMA Series) 64
3.8.6 PCLMA (EFL-PCLMA Series) 64
References 66
4 Coaxial 3D Bioprinting69
4.1 Introduction 69
4.1.1 Significance 69
4.1.2 Two Categories 72
4.1.2.1 Solid Fiber-Based Coaxial Bioprinting 72
4.1.2.2 Hollow Fiber-Based Coaxial Bioprinting 73
4.2 Printable Ink Materials 74
4.2.1 Forming Mechanism 74
4.2.2 Categories of Printable Bioinks 75
4.2.2.1 Alginate 75
4.2.2.2 Gelatin 78
4.2.2.3 GelMA 79
4.3 Representative Biomedical Applications 80
4.3.1 Morphology-Controllable Microfiber-Based Organoids 80
4.3.2 Vessel-on-a-Chip 81
4.4 Future Perspective 85
References 86
5 Digital Light Projection-Based 3D Bioprinting89
5.1 Introduction 89
5.1.1 Printing Process 89
5.1.2 Significance 89
5.2 Photocurable Biomaterials 91
5.2.1 Photo-Cross-Linking Mechanism 92
5.2.1.1 Conversion of Light Energy to Chemical Energy: Photoinitiator 92
5.2.1.2 Formation of Molecular Network: Monomer Polymerization 93
5.2.2 Typical Materials: Gelatin Methacryloyl (GelMA) 94
5.2.2.1 Composition and Synthesis 94
5.2.2.2 Substitution Degree 95
5.3 Printing Equipment 96
5.3.1 Optical Units 96
5.3.1.1 Image Forming: Digital Micromirror Devices 97
5.3.1.2 Objective Lens: Focusing System 97
5.3.1.3 Material Storage Units 98
5.3.1.4 Environment Controlling Systems 98
5.3.1.5 Ink Tank: Transparent and Non-stick Bottom 99
5.4 Mechanical Movement Units 99
5.4.1 Lifting Mechanism: Main Movement 99
5.4.2 Tilting Mechanism: Mixing and Separation 100
5.4.2.1 Printing Error Formation and Optimization Strategies 100
5.5 Optimization of Several Typical Structures 102
5.5.1 Printing Strategies of Solid Structures 103
5.5.2 Printing Strategies of Channel Structures 104
5.5.3 Printing Strategies of Conduit Structures 104
5.5.4 Printing Strategies of Thin-Walled Structures 105
5.5.5 Printing Strategies of Microcolumn Structures 105
5.6 Applications 107
5.6.1 DLPBP Structures with High Precision 107
5.6.2 Customized Physical Properties Bioprinting 107
5.6.3 Regenerative and Biomedical Applications 108
References 110
6 Direct Ink Writing for 3D Bioprinting Applications113
6.1 Introduction 113
6.2 Printable Bioinks in DIW 114
6.2.1 Supporting Mechanisms and Representative Bioinks 115
6.2.1.1 Rapid Solidification-Induced Mechanical Stiffness Improvement 115
6.2.1.2 Yield-stress Additive-Induced Self-Supporting Capacity 119
6.2.2 Design Criteria of Bioinks for Direct Writing Applications 121
6.2.2.1 Rheological Properties 122
6.2.2.2 Cross-linking Capacity 122
6.2.2.3 Biocompatibility and Biodegradation 123
6.2.2.4 Mechanical Properties 124
6.3 Technical Specifics in Direct Ink Writing 124
6.3.1 Investigation on Printability of Bioinks 124
6.3.2 Different Printing Strategies in Rapid Solidification-Induced 3D Printing Approach 126
6.3.2.1 Printing of Thermal Cross-linkable Biomaterials 126
6.3.2.2 Printing of Ionic Cross-linkable Biomaterials 127
6.3.2.3 Printing of Photo Cross-linkable Biomaterials 128
6.3.2.4 Printing of Enzyme Cross-linkable Biomaterials 129
6.3.3 3D Structure Printing Using Self-Supporting Material-Assisted 3D Printing Approach 130
6.3.3.1 Internal Scaffold Additive-Assisted 3D Printing 130
6.3.3.2 Microgel Additive-Assisted 3D Printing 132
6.4 Representative Biomedical Applications 132
6.4.1 Aortic Valve Printing 132
6.4.2 Bone and Cartilage Tissue Printing 133
6.4.3 Cardiac Tissue Printing 134
6.4.4 Liver Tissue Printing 135
6.4.5 Lung Tissue Printing 135
6.4.6 Neural Tissue Printing 135
6.4.7 Eye and Ear Printing 136
6.4.8 Pancreas Printing 137
6.4.9 Skin Tissue Printing 137
6.4.10 Blood Vessel Printing 138
6.5 Conclusions and Future Work 138
References 139
7 Liquid Support BathAssisted 3D Bioprinting149
7.1 Introduction 149
7.2 Liquid Support Bath Materials 150
7.2.1 Support Bath Materials Based on Different Supporting Mechanisms 151
7.2.1.1 Unrecoverable Matrix Materials 151
7.2.1.2 Buoyant Support Fluids 151
7.2.1.3 Reversibly Self-Healing Hydrogels 153
7.2.1.4 Yield-Stress Fluids 154
7.2.2 Preparation Methods 156
7.2.2.1 Microparticle Aggregation 156
7.2.2.2 Homogenous Suspensions with Micro/Nanostructures 157
7.2.2.3 Chemical Synthesis 158
7.2.2.4 Other Methods 158
7.2.3 Design Criteria for Ideal Liquid Support Bath Material 158
7.2.3.1 Rheological Properties 158
7.2.3.2 Chemical Stability 159
7.2.3.3 Physical Stability 159
7.2.3.4 Biocompatibility 161
7.2.3.5 Hydrophilicity and Hydrophobicity 161
7.2.3.6 Others 161
7.3 Scientific Issues During Liquid Support BathAssisted 3D Printing 162
7.3.1 Effects of Operating Conditions on Filament Formation in Support Bath 162
7.3.2 Effects of Support Bath Materials on Filament Morphology 162
7.3.2.1 Rheological Properties of Support Bath Materials 162
7.3.2.2 Diffusion of Ink Materials into Surrounding Support Bath 163
7.3.2.3 Interfacial TensionInduced Filament Deformation 165
7.3.3 Effects of Nozzle Movement on the Printed Structure 165
7.3.4 Path Design in Liquid Support BathAssisted 3D Printing 166
7.4 Post-treatments for Liquid Support BathAssisted 3D Printing 167
7.4.1 Post-treatments in e-3DP 167
7.4.2 Post-treatments in Support BathEnabled 3D Printing 169
7.5 Representative Biomedical Applications 169
7.5.1 Organ Printing 169
7.5.2 Lab-on-a-Chip 171
7.5.3 Other Bio-Related Applications 173
7.6 Conclusions and Future Directions 173
References 175
8 Bioprinting Approaches of Hydrogel Microgel179
8.1 Introduction 179
8.2 Auxiliary Dripping 179
8.2.1 Inkjet Printing 180
8.2.1.1 Piezoelectric Inkjet 180
8.2.1.2 Thermal Bubble Inkjet 183
8.2.2 Laser-Assisted Printing 184
8.2.3 Electrohydrodynamic Printing 185
8.3 Diphase Emulsion 195
8.3.1 Nonaqueous Liquid Stirring 195
8.3.2 Air-Assisted Atomization 197
8.3.3 Microfluidic Technology 198
8.4 Lithography Technology 202
8.4.1 Replica Mold 202
8.4.2 Discrepant Wettability 203
8.4.3 Photomask Film 206
8.4.4 Digital Light Processing 208
8.5 Bulk Crushing 208
References 211
9 Biomedical Applications of Microgels213
9.1 Introduction 213
9.1.1 Tiny Size 213
9.1.2 Hydrogel Network 213
9.1.3 Complex Mechanical Properties 214
9.2 In Vitro Model 214
9.3 Cell Therapy 216
9.4 Drug Delivery 219
9.5 Cell Amplification 223
9.6 Single-Cell Capture 227
9.7 Supporting Matrices 229
9.8 Secondary Bioprinting 232
References 235
10 Microfiber-Based Organoids Bioprinting for In Vitro Model237
10.1 Introduction 237
10.1.1 Significance and Challenge 237
10.1.2 Hydrogel Materials 238
10.2 Coaxial Bioprinting of Bioactive Cell-laden Microfiber 238
10.2.1 Microfluidic Coaxial Bioprinting 239
10.2.2 Coaxial Nozzle-Assisted Bioprinting 240
10.3 Heteromorphic/Heterogeneous Microfiber Bioprinting 241
10.3.1 Heteromorphic Microfiber 242
10.3.2 Heterogeneous Microfiber 244
10.4 3D Assembly of Microfibers 245
10.4.1 3D Bioweaving 245
10.4.2 3D Bioprinting 245
10.5 Microfiber-Based Organoids Bioprinting for In Vitro Mini Tissue Models 247
10.5.1 Vascular Organoid 247
10.5.2 Myocyte Fiber 248
10.5.3 Nerve Fiber 248
10.5.4 Cardiomyocyte Fiber 249
10.5.5 Co-cultured Multi-organoids Interactions 249
10.6 Discussion and Outlook 250
References 251
11 Large Scale Tissues Bioprinting257
11.1 Introduction 257
11.1.1 Challenges in Bioprinting Large Scale Tissues 257
11.1.2 Strategies in Bioprinting Large Scale Tissue with Nutrient Networks 258
11.1.2.1 Porous Network Printing 258
11.1.2.2 Hollow Channel Network Printing 259
11.1.2.3 Advanced Bioprinting Techniques-Enabled Printing Highly Biomimetic Vascular Network 259
11.2 Large Scale Cell-laden Porous Structures Printing 259
11.2.1 Independent Porous Structure Printing 259
11.2.2 Interconnected Porous Structure Printing 261
11.2.2.1 Directly Cell-laden Scaffold Printing 261
11.2.2.2 Synchronous Bioprinting (Bioink and Sacrificial Ink Half and Half) 261
11.2.3 Heterogeneous Independent/Interconnected Porous Structure Printing 262
11.2.4 Long-term Perfusion Culture on a Chip 265
11.2.5 Discussions (Properties, Pros, Cons, etc.) 265
11.3 Large Scale Cell-laden Structures with Vascular Channel Printing 266
11.3.1 Sacrificial Bioprinting 266
11.3.2 Coaxial Bioprinting 267
11.4 One-step Coaxial/Sacrificial Printing of Large Scale Vascularized Tissue Constructs 268
11.4.1 Mechanism 268
11.4.2 Freeform Structure with Vascular Channels Printing 269
11.4.3 Heterogeneous Structure with Vascular Channels Printing 270
11.4.4 Long-term Perfusion Culture on a Chip 272
11.4.5 Discussion (Properties, Pros and Cons, etc.) 272
11.5 Advanced Bioprinting Technique-Enabled Printing Highly Biomimetic Tissues 273
11.5.1 Support Bath-Assisted Bioprinting 273
11.5.2 Light-Based Bioprinting 273
11.5.3 Discussion (Properties, Pros and Cons, etc.) 275
11.6 Representative Biomedical Applications 275
References 276
12 3D Printing of Vascular Chips281
12.1 Introduction 281
12.2 Construction Process of Hydrogel-Based Vascular Chips 282
12.2.1 Damage-Free Demolding Process Based on Soft Fiber Template 282
12.2.1.1 Damage-Free Demolding Process 283
12.2.1.2 Comparative Analysis of Damage-Free and Conventional Demolding Processes 283
12.2.2 Hydrogel Bonding Strategy Based on Twice-Cross-linking Mechanism 286
12.2.2.1 Manufacturing Process of Hydrogel-Based Microfluidic Chips 287
12.2.2.2 Mechanism Study 287
12.2.2.3 Material Selection 288
12.2.2.4 Feasible Domain 289
12.2.2.5 Bonding Results 289
12.2.3 Multi-Scale 3D Printing Process 291
12.2.3.1 Mechanism of Multi-Scale 3D Printing Process 291
12.2.3.2 Printing Parameters 292
12.2.4 Construction Process of Hydrogel-Based Vascular Chips 293
12.3 Characterization of Vascular Chips 295
12.3.1 Fundamental Characterization of Vascular Chips 295
12.3.1.1 Characterization of Endothelium Function of Channels 295
12.3.1.2 Characterization of Endothelial Cells Viability 295
12.3.1.3 Characterization of Endothelial Cells Morphology 296
12.3.1.4 Characterization of Endothelium Channel 297
12.3.2 Morphology Characterization of Hydrogel-Based Vascular Chips 298
12.3.2.1 Multi-Level Bifurcated Channel Network Structure 298
12.3.2.2 Multi-Scale Vascular Model 299
12.3.2.3 Biomimicking Vascular Model 299
12.3.3 Characterization of Vascular Function 302
12.3.3.1 Nutrition Supply Function 302
12.3.3.2 Expression of Key Functional Proteins in Endothelial Cells 302
12.3.3.3 Simulation of Vascular Inflammation Reaction 303
12.3.3.4 Characterization of Vascular Barrier Function 304
12.4 Conclusion 307
References 308
13 3D Printing of In Vitro Models311
13.1 Introduction 311
13.2 Typical 3D Bioprinting Technologies and Common Target Tissue/Organ Demand 312
13.2.1 Inkjet-Based Bioprinting 313
13.2.2 Extrusion-Based Bioprinting 314
13.2.3 Light-Assisted Bioprinting 315
13.3 Developing Process of In Vitro Models 316
13.3.1 Mini-Tissue in 3D Growth State 316
13.3.1.1 Sphere Mini-Tissue Model 316
13.3.1.2 Fiber Mini-Tissue Model 317
13.3.1.3 Array Mini-Tissue Model 318
13.3.1.4 Limitations 319
13.3.2 Organ-on-a-Chip with Multiplex Microenvironment 319
13.3.2.1 Integrated Organ-on-a-Chip 321
13.3.2.2 Modular Microfluidic System 322
13.3.2.3 Multiple-Organ System 323
13.3.2.4 Limitations 325
13.3.3 Tissue/Organ Construct with Biomimicking Property 325
13.3.3.1 Vascular Construct 326
13.3.3.2 Vascularized Tissue Construct 328
13.3.3.3 Limitations 330
13.4 3D Printing of In Vitro Tumor Models 330
13.4.1 Tumor Cell-Laden Construct 330
13.4.2 Multi-Cell Tumor Sphere 331
13.4.3 Tumor Metastasis Model with Angiogenesis 332
13.5 Summary and Prospect 334
13.5.1 Key Virtue and Comparison 334
13.5.2 Outlook 334
13.5.2.1 3D Bioprinting Technology 335
13.5.2.2 Individual Differences 335
13.5.2.3 Systematic Interaction 335
13.5.2.4 Industrialization 335
13.6 Conclusions 336
References 336
14 Protocol of Typical 3D Bioprinting 339
Reference 343
Index 345
