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- Herausgeber: Wiley-VCH GmbH
- Kategorie: Fachliteratur
- Sprache: Englisch
Provides an up-to-date outline of cell assembly methods and applications of 3D bioprinting
Cell Assembly with 3D Bioprinting provides an accesible overview of the layer-by-layer manufacturing of living structures using biomaterials. Focusing on technical implemention in medical and bioengineering applications, this practical guide summarize each key aspect of the 3D bioprinting process. Contributions from a team of leading researchers describe bioink preparation, printing method selection, experimental protocols, integration with specific applications, and more.
Detailed, highly illustrated chapters cover different bioprinting approaches and their applications, including coaxial bioprinting, digital light projection, direct ink writing, liquid support bath-assisted 3D printing, and microgel-, microfiber-, and microfluidics-based biofabrication. The book includes practical examples of 3D bioprinting, a protocol for typical 3D bioprinting, and relevant experimental data drawn from recent research.
* Highlights the interdisciplinary nature of 3D bioprinting and its applications in biology, medicine, and pharmaceutical science
* Summarizes a variety of commonly used 3D bioprinting methods
* Describes the design and preparation of various types of bioinks
* Discusses applications of 3D bioprinting such as organ development, toxicological research, clinical transplantation, and tissue repair
Covering a wide range of topics, Cell Assembly with 3D Bioprinting is essential reading for advanced students, academic researchers, and industry professionals in fields including biomedicine, tissue engineering, bioengineering, drug development, pharmacology, bioglogical screening, and mechanical engineering.
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Veröffentlichungsjahr: 2021
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Table of Contents
Cover
Title Page
Copyright
Preface
1 3D Bioprinting, A Powerful Tool for 3D Cells Assembling
1.1 What Is 3D Bioprinting?
1.2 Evolution of 3D Bioprinting
1.3 Brief Classification of 3D Bioprinting
1.4 Evaluation of Bioinks
1.5 Outlook and Discussion
References
2 Representative 3D Bioprinting Approaches
2.1 Introduction
2.2 Inkjet Bioprinting
2.3 Extrusion Bioprinting
2.4 Light‐Based Bioprinting
References
3 Bioink Design: From Shape to Function
3.1 Significance of Bioink Design
3.2 Categories of Bioink
3.3 Three Evaluation Criteria of Bioink
3.4 Strategies for Enabling the Printability
3.5 Strategies for Bioink Reinforcement
3.6 Strategies for Improving the Biocompatibility
3.7 Representative Bioink Design Case: GelMA‐Based Bioinks
3.8 Commercial Bioink
References
4 Coaxial 3D Bioprinting
4.1 Introduction
4.2 Printable Ink Materials
4.3 Representative Biomedical Applications
4.4 Future Perspective
References
5 Digital Light Projection‐Based 3D Bioprinting
5.1 Introduction
5.2 Photocurable Biomaterials
5.3 Printing Equipment
5.4 Mechanical Movement Units
5.5 Optimization of Several Typical Structures
5.6 Applications
References
6 Direct Ink Writing for 3D Bioprinting Applications
6.1 Introduction
6.2 Printable Bioinks in DIW
6.3 Technical Specifics in Direct Ink Writing
6.4 Representative Biomedical Applications
6.5 Conclusions and Future Work
References
7 Liquid Support Bath–Assisted 3D Bioprinting
7.1 Introduction
7.2 Liquid Support Bath Materials
7.3 Scientific Issues During Liquid Support Bath–Assisted 3D Printing
7.4 Post‐treatments for Liquid Support Bath–Assisted 3D Printing
7.5 Representative Biomedical Applications
7.6 Conclusions and Future Directions
References
8 Bioprinting Approaches of Hydrogel Microgel
8.1 Introduction
8.2 Auxiliary Dripping
8.3 Diphase Emulsion
8.4 Lithography Technology
8.5 Bulk Crushing
References
9 Biomedical Applications of Microgels
9.1 Introduction
9.2 In Vitro Model
9.3 Cell Therapy
9.4 Drug Delivery
9.5 Cell Amplification
9.6 Single‐Cell Capture
9.7 Supporting Matrices
9.8 Secondary Bioprinting
References
10 Microfiber‐Based Organoids Bioprinting for In Vitro Model
10.1 Introduction
10.2 Coaxial Bioprinting of Bioactive Cell‐laden Microfiber
10.3 Heteromorphic/Heterogeneous Microfiber Bioprinting
10.4 3D Assembly of Microfibers
10.5 Microfiber‐Based Organoids Bioprinting for In Vitro Mini Tissue Models
10.6 Discussion and Outlook
References
11 Large Scale Tissues Bioprinting
11.1 Introduction
11.2 Large Scale Cell‐laden Porous Structures Printing
11.3 Large Scale Cell‐laden Structures with Vascular Channel Printing
11.4 One‐step Coaxial/Sacrificial Printing of Large Scale Vascularized Tissue Constructs
11.5 Advanced Bioprinting Technique‐Enabled Printing Highly Biomimetic Tissues
11.6 Representative Biomedical Applications
References
12 3D Printing of Vascular Chips
12.1 Introduction
12.2 Construction Process of Hydrogel‐Based Vascular Chips
12.3 Characterization of Vascular Chips
12.4 Conclusion
References
13 3D Printing of In Vitro Models
13.1 Introduction
13.2 Typical 3D Bioprinting Technologies and Common Target Tissue/Organ Demand
13.3 Developing Process of In Vitro Models
13.4 3D Printing of In Vitro Tumor Models
13.5 Summary and Prospect
13.6 Conclusions
References
14 Protocol of Typical 3D Bioprinting
Reference
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 Timeline for bioprinting evolution.
Chapter 2
Table 2.1 Hydrogels applied for inkjet bioprinting of 3D constructs.
Chapter 3
Table 3.1 Representative cell type that can be cultured with GelMA.
Table 3.2 Photo‐cross‐linking bioink of EFL.
Chapter 4
Table 4.1 Different bioinks in coaxial bioprinting.
Chapter 5
Table 5.1 How to change these parameters to improve prints.
Table 5.2 The selections of parameters in each printing strategy.
Table 5.3 The summary sheet of printing strategies.
Chapter 7
Table 7.1 Summary of critical properties of commonly used liquid support b...
Table 7.2 Post‐treatments for liquid support bath–assisted 3D printing.
Chapter 13
Table 13.1 Advantages and disadvantages of the in vitro methods for drug s...
List of Illustrations
Chapter 1
Figure 1.1 Introduction of 3D bioprinting.
Chapter 2
Figure 2.1 Classification of representative 3D bioprinting approaches.
Figure 2.2 Mechanisms of droplet formation in inkjet bioprinting. (a) Contin...
Figure 2.3 Representative inkjet bioprinting results based on different drop...
Figure 2.4 Representative biomedical applications of inkjet bioprinting. (a)...
Figure 2.5 Different mechanisms for biomaterials deposition. (a) High pressu...
Figure 2.6 Two main extrusion bioprinting strategies: (a) direct ink writing...
Figure 2.7 Extrusion bioprinting of complex 3D structures from different typ...
Figure 2.8 (a) Mechanism of laser‐assisted bioprinting without and with EAL....
Figure 2.9 Representative biomedical applications of laser‐assisted bioprint...
Figure 2.10 (a) Schematic of stereolithography bioprinting.
Figure 2.11 (a) Mechanism of MPP bioprinting.
Figure 2.12 (a) Mechanism of digital light projection 3D printing.
Figure 2.13 (a) Mechanism of computed axial lithography‐based printing....
Chapter 3
Figure 3.1 (a) Multilevel scale pore and respective functions in tissue engi...
Figure 3.2 (a) The mechanism of hydrogel reinforcement with organized high‐p...
Figure 3.3 (a) The synthesis principle of GelMA. (b) The nuclear magnetic sp...
Figure 3.4 The effect of substitution rate and concentration on the viscosit...
Figure 3.5 The effect of substitution rates and concentrations on the sol–ge...
Figure 3.6 The effect of substitution rates and concentrations on the photo‐...
Figure 3.7 The effect of substitution rates and concentrations on compressio...
Figure 3.8 (a) The growth state of BMSCs encapsulated within GelMA. (b) The ...
Figure 3.9 (a) Cross‐linking steps in the process of bioprinting GelMA bioin...
Figure 3.10 (a) 3D printing strategy of complex scaffolds with GelMA/nanocla...
Figure 3.11 Information of GelMA (EFL‐GM series). (a) Product type. (b) Prin...
Figure 3.12 Information of fluorescent GelMA (EFL‐GM‐F series). (a) Product ...
Figure 3.13 Information of porous GelMA (EFL‐GM‐PR series). (a) Product type...
Figure 3.14 Information of porous HAMA (EFL‐HAMA series). (a) Product type. ...
Figure 3.15 Information of SilMA (EFL‐SilMA series). (a) Product type. (b) P...
Figure 3.16 Information of PCLMA (EFL‐PCLMA series). (a) Product type. (b) P...
Chapter 4
Figure 4.1 Requirements to fabricate vascular organ prototypes by coaxial bi...
Figure 4.2 Schematic illustration of fabrication method of coaxial bioprinti...
Figure 4.3 Schematic illustration of current progress of morphology‐controll...
Figure 4.4 Schematic illustration of current progress of vessel‐on‐a‐chip by...
Chapter 5
Figure 5.1 Complex DLP printed models with high precision, superior biocompa...
Figure 5.2 The process that lithium acylphosphinate (LAP) initiated by 405 n...
Figure 5.3 In the process of forming a cross‐linked molecular network, there...
Figure 5.4 The reaction principle of using gelatin and MA to synthesize GelM...
Figure 5.5
1
H NMR spectroscopy of gelatin and GelMA, and characteristic peaks...
Figure 5.6 The structure of digital micromirror device: how to form a digita...
Figure 5.7 The multilayer structure of transparent ink tank bottom.
Figure 5.8 Main movement: platform rising and going down.
Figure 5.9 Tilting mechanism: mixing and separation.
Figure 5.10 The principle of forming printing errors in the
z
‐direction.
Figure 5.11 The principle of forming printing errors in the
x
–
y
plane.
Figure 5.12 Five types of structures proposed from specific applications.
Figure 5.13 DLPBP GelMA models: ear, rose, and lotus.
Figure 5.14 Mechanical parameter map: controllable mechanical properties....
Figure 5.15 DLPBP application case: mechanical properties controllable and b...
Chapter 6
Figure 6.1 Schematic of (a) rapid solidification‐induced 3D bioprinting stra...
Figure 6.2 Classification of cross‐linking strategies during rapid solidific...
Figure 6.3 Engineered cell sheet using pNIPAAm‐grafted surfaces. (a) Schemat...
Figure 6.4 (a) Inherent “house‐of‐cards” arrangement of Laponite® nanoclay s...
Figure 6.5 Schematic of microgel additive‐assisted 3D bioprinting strategy. ...
Figure 6.6 Assessment of the bioink printability. (a) Different types of fil...
Figure 6.7 Strategies for thermal cross‐linkable bioink printing. (a) Heatin...
Figure 6.8 Different strategies for 3D printing of ionic cross‐linkable bioi...
Figure 6.9 Three strategies for 3D printing of photo cross‐linkable bioinks....
Figure 6.10 Printing‐mixing system for 3D printing of enzyme cross‐linkable ...
Figure 6.11 3D structures printed using self‐supporting material‐assisted 3D...
Figure 6.12 3D bioprinting of aortic valves using DIW. (a) 3D printed axisym...
Figure 6.13 (a) 3D printed PCL‐PLGA‐TCP scaffolds with mesenchymal stromal c...
Figure 6.14 (a) Schematic of the timeline for bioprinting the two cell‐layer...
Figure 6.15 (a) A schematic describing the approach by which amniotic fluid‐...
Chapter 7
Figure 7.1 Liquid support bath–assisted 3D bioprinting techniques. (a) 3D li...
Figure 7.2 (a–e) Schematics of omnidirectional printing of 3D microvascular ...
Figure 7.3 Schematic diagram of printing in a buoyant fluid bath.
Figure 7.4 (a) Schematic diagram of self‐healing hydrogels and self‐healing ...
Figure 7.5 Schematics of printing mechanisms using (a) granular gel medium....
Figure 7.6 Formation of microgel particles using the microfluidic system....
Figure 7.7 (a) Schematic diagram of filament printing in a liquid support ba...
Figure 7.8 (a) PDMS filament printing in different Carbopol support baths....
Figure 7.9 (a) Dispensing nozzle moving in a support bath of yield‐stress fl...
Figure 7.10 Localized “layer‐by‐layer” path design strategy.
Figure 7.11 Representative applications in organ printing. (a) (a‐1) Vascula...
Figure 7.12 Representative lab‐on‐a‐chip applications. (a) Heterogeneous cel...
Figure 7.13 Representative bio‐related applications via liquid support bath–...
Chapter 8
Figure 8.1 (a) Schematic diagram showing the experimental setup. (b) Bipolar...
Figure 8.2 Formed alginate microparticles with a combination of the microdro...
Figure 8.3 Alginate microspheres embedded with living cells.
Figure 8.4 The setup used for the preparation of the alginate gel microspher...
Figure 8.5 Laser printing schematic with an inset illustrating a printing pr...
Figure 8.6 Sketch of the electro‐assisted printing device, fabricating proce...
Figure 8.7 The analysis of different printing states. (a) The diagrammatic s...
Figure 8.8 Cellular encapsulation. (a) Confocal images of LIVE/DEAD staining...
Figure 8.9 Drug‐controlled release. (a) The confocal fluorescent microscopy ...
Figure 8.10 The sketch of the train of thought and fabrication process. (a) ...
Figure 8.11 3D spiral‐based droplet formation from airflow‐driven rotation. ...
Figure 8.12 (a–c) Spheroids with spiral‐compartmental geometries with spiral...
Figure 8.13 Organoids construction through the coculture of HUVECS and HMSCs...
Figure 8.14 Principle of visual positioning of the airflow needle.
Figure 8.15 Mathematic analysis of the spinning process of microspheroids. (...
Figure 8.16 Inverse suspension polymerization of microgel building blocks. S...
Figure 8.17 Diagram of vortex‐induced emulsion encapsulation method. A polym...
Figure 8.18 Scheme of the cell‐spraying device: (1) a cell‐laden alginate sp...
Figure 8.19 One‐step production of microgels by the use of double emulsion d...
Figure 8.20 Capillary microfluidic device and the formation of precisely con...
Figure 8.21 Schematic representation of degassed micromolding lithography. (...
Figure 8.22 Surface‐wettability‐guided assembly of microgel arrays. (a) Sche...
Figure 8.23 Hydrogel array formation procedure and outputs. (a and b) Hydrog...
Figure 8.24 Schematic representation of the hydrogel assembly process. Indiv...
Figure 8.25 Scanning electron micrographs of PEG‐DA (MW 575) gel microstruct...
Figure 8.26 (a) Schematic representation of DLP‐3D printer for microgels cus...
Figure 8.27 Production, properties, and applications of “ZIP” hydrogels. (a)...
Figure 8.28 3D bioprinting large‐scale tissue constructs with MPNs. (a) Sche...
Chapter 9
Figure 9.1 Schematic of fabrication processes for PEG‐Fb tumor microsphere (...
Figure 9.2 Sketch of the pioneering strategy. (a) Inspiration of this strate...
Figure 9.3 Schematic diagram of fabrication of BMSC‐laden GelMA microspheres...
Figure 9.4 Bone defect repair in vivo. (a) Operation processes. (i) Schemati...
Figure 9.5 Generated microspheres containing VEGF‐overexpressing HEK293T cel...
Figure 9.6 3D‐printed concentration‐controlled microfluidic chip for Dox del...
Figure 9.7 OVA encapsulated core–shell alginate microparticles coated with c...
Figure 9.8 Summary of this study and schematic diagram of 3D DLP technology....
Figure 9.9 The printed GelMA MSs for cell passage, cell harvest, and cell cr...
Figure 9.10 (a)–(c) The fluorescent images of the overall HUMSC‐seeded GCS/G...
Figure 9.11 HUMSCs cytoskeleton and stromal markers immunofluorescence stain...
Figure 9.12 (a) Schematic diagram of the DLMF for the separation of CTCs. Th...
Figure 9.13 Shear‐thinning and self‐healing alginate microgel supporting med...
Figure 9.14 Differentiation of 3D bioprinted hMSC constructs. (a) Digital im...
Figure 9.15 The design and preparation of a multiscale composite scaffold mi...
Figure 9.16 The bioprinting of the multiscale GC–MS+NGF/GelMA composite scaf...
Chapter 10
Figure 10.1 Coaxial laminar flow microfluidic device (a); core–shell cell‐la...
Figure 10.2 (a) Mechanism of coaxial nozzle‐assisted bioprinting.
Figure 10.3 The mechanism of fabricating morphology‐controllable GelMA micro...
Figure 10.4 With increase of the flow rate ratio (
Q
in
/
Q
out
), the morphology ...
Figure 10.5 Two‐in‐one coaxial nozzle, three‐layer coaxial nozzle and one pa...
Figure 10.6 (a) Different proportions of Janus GelMA microfibers.
Figure 10.7 3D bioweaving of CLHMs or MFOs.
Figure 10.8 3D bioprinting of CLHMs. 3D heterogeneous constructs and 3D heli...
Figure 10.9 Microfluidic coaxial bioprinting of vascular organoids.
Figure 10.10 Coaxial nozzle‐assisted bioprinting of vascular organoids.
Figure 10.11 Microfluidic coaxial bioprinting of myocyte fiber.
Figure 10.12 Microfluidic coaxial bioprinting of nerve fiber. GFAP, magenta;...
Figure 10.13 Microfluidic coaxial bioprinting of cardiomyocyte fiber.
Figure 10.14 Co‐cultured multi‐organoids interactions by CLHMs. Angiogenic s...
Chapter 11
Figure 11.1 (a) The preparation of sacrificial gelatin microgel‐laden GelMA ...
Figure 11.2 Direct bioprinting of cell‐laden scaffold. Due to the insufficie...
Figure 11.3 (a) Synchronous bioprinting strategy and specific steps.
Figure 11.4 Heterogeneous 3D bioprinting of independent pore structure throu...
Figure 11.5 Heterogeneous 3D bioprinting of interconnected pore structure th...
Figure 11.6 Sacrificial bioprinting of vascular channels.
Figure 11.7 Coaxial bioprinting of vascular channels.
Figure 11.8 (a) Mechanism of one‐step coaxial/sacrificial printing strategy ...
Figure 11.9 Based on the above one‐step coaxial/sacrificial printing strateg...
Figure 11.10 Bioprinting multicomponent/multicellular tissue constructs thro...
Figure 11.11 Support bath‐assisted bioprinting of high biomimetic tissue str...
Figure 11.12 Light‐based bioprinting of high biomimetic tissue structures co...
Chapter 12
Figure 12.1 Fabrication of the hydrogel micro structure: (a) 3D printing of ...
Figure 12.2 Preliminary comparison of damage‐free demolding and the conventi...
Figure 12.3 Mechanical analysis of two demolding processes including the mic...
Figure 12.4 Schematic of the fabrication process for the hydrogel‐based chip...
Figure 12.5 Mechanism of twice‐cross‐linking bonding strategy.
Figure 12.6 Images of cell attachment and spreading for the three composite ...
Figure 12.7 The feasible domain for stripping (a) and bonding (b) processes....
Figure 12.8 Cross‐section images of channels: rectangular channel (a) and ci...
Figure 12.9 The tensile curves for both casting and bonding hydrogels.
Figure 12.10 Printing principle of the multi‐scale template.
Figure 12.11 Statistical analysis of the processing parameters during the EH...
Figure 12.12 Quantitative analysis of the various channel diameters during t...
Figure 12.13 Schematic for the construction of a multi‐scale vascular chip....
Figure 12.14 Photographs of 300 and 500 μm microchannels with
human umbilica
...
Figure 12.15 Schematic for the endothelialization process of HUVECs along wi...
Figure 12.16 Confocal images of LIVE/DEAD staining of the endothelialized ch...
Figure 12.17 Confocal images of cytoskeleton staining of the endothelialized...
Figure 12.18 3D stacking confocal images of the
endothelial cell
(
EC
) channe...
Figure 12.19 (a) Microscopy morphology of the longitudinal section of the EC...
Figure 12.20 (a)
Scanning electron microscope
(
SEM
) image of the inner wall ...
Figure 12.21
In vivo
vascular system.
Figure 12.22 Display of the multi‐scale channel network and EC channels with...
Figure 12.23 Fluorescence micrograph of a three‐level EC network.
Figure 12.24 Fluorescence micrograph of a spiral EC channel.
Figure 12.25 Fluorescence micrograph of a stenosis‐mimicking EC channel.
Figure 12.26 (a) Viability of the tumor cells in the model. (b) Cell prolife...
Figure 12.27 Fluorescence micrograph of VE‐cadherin/CD31/vinculin markers of...
Figure 12.28 (a) The
messenger RNA
(
mRNA
) levels of ICAM‐1 in ECs. (b) The m...
Figure 12.29 Presentation of the empty channels and the EC channels for both...
Figure 12.30 Schematic for the endothelialization process of HUVECs along wi...
Figure 12.31 Time‐lapse confocal images of the diffusional permeability patt...
Figure 12.32 Diffusional permeability calculation of dextran.
Figure 12.33 Time‐lapse fluorescence images of diffusion patterns of 10 and ...
Figure 12.34 Line plots of fluorescence across the channel after 30 minutes ...
Chapter 13
Figure 13.1 Schematic diagram displaying the routing of drug development. Dr...
Figure 13.2 Inkjet‐based bioprinting systems.
Figure 13.3 Extrusion‐based bioprinting systems.
Figure 13.4 Light‐assisted bioprinting systems.
Figure 13.5 Sphere‐shaped mini‐tissue: (a) GelMA microspheres.
Figure 13.6 Fiber‐shaped mini‐tissue: (a) Straight and spiral fibers, vessel...
Figure 13.7 Mini‐tissue array: (a) Cell‐laden mini‐tissue array.
Figure 13.8 Integrated cell/organ‐on‐a‐chip: (a) Schematic diagram of the in...
Figure 13.9 Modular microfluidic system: (a) Discrete elements with standard...
Figure 13.10 Multiple‐organ system: (a) Schematic diagram of a human‐on‐a‐ch...
Figure 13.11 Vascular constructs: (a) Schematic diagram of the construction ...
Figure 13.12 Vascularized tissue constructs: (a) Schematic of the fabricatio...
Figure 13.13 Schematic of the printing process and images of 3D HeLa cell‐la...
Figure 13.14 Schematic of drug cytotoxicity and signaling pathway analysis w...
Figure 13.15 Tumor metastasis model: (a) Schematic of the tumor development ...
Chapter 14
Figure 14.1 Bioprinting sketch of GelMA microspheres.
Figure 14.2 Bioprinting sketch of GelMA fibers.
Figure 14.3 Bioprinting sketch of complex 3D GelMA structures.
Figure 14.4 Fabrication sketch of GelMA‐based microfluidic chips.
Guide
Cover Page
Table of Contents
Title Page
Copyright
Preface
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Cell Assembly with 3D Bioprinting
Yong HeQing GaoYifei Jin
Authors
Prof. Yong He
School of Mechanical Engineering
Room 123, Teaching Building 1,
Yuquan Campus, Zhejiang University
No. 38, Zheda Road
310027 Hangzhou
China
Dr. Qing Gao
School of Mechanical Engineering
Room 123, Teaching Building 1,
Yuquan Campus, Zhejiang University
No. 38, Zheda Road
310027 Hangzhou
China
Prof. Yifei Jin
University of Nevada, Reno
Department of Mechanical Engineering
Room 230
1664 N. Virginia Street
NV
United States
Cover Image: Yong He
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Cover Design Adam‐Design, Weinheim, Germany
Preface
3D bioprinting has opened up a frontier in biomedical research, aiming at additive manufacturing or assembling living structures with cells, which provides the possibility to generate significant breakthroughs, yielding new treatments, and change the foundation of regenerative medicine. It is truly an interdisciplinary field, cross‐fertilized ranging from mechanical engineering, materials science, and computer science to biology, medicine, pharmaceutical science, and so on.
The potential applications for 3D bioprinting include: (i) in vitro 3D tissue/organ models for drug screening, organ development, toxicological and cosmetic research, etc.; (ii) 3D biofabrication of living structures for clinical transplantation or tissue repair.
Compared to conventional additive manufacturing, 3D bioprinting possesses three remarkable characteristics: (i) bioprinting usually utilizes cell‐laden hydrogel (called bioink) in terms of material use; (ii) bioprinted structures have to go through hydrogel crosslinking process (thermal, chemical, or enzymatic) during formation for manufacturing desired tissue structures; (iii) cross‐talking and functionalization of cells to acquire some tissue properties after printing is the goal. In these regards, bioprinting faces two major challenges: (i) structural controlled manufacturing, which requires a stable printing process to ensure cell‐laden hydrogels being accurately assembled. As the bioink is something like a soft tofu, precision manufacturing is difficult; (ii) functional controlled postprocessing, which needs to provide biomimetic microenvironment with physical and chemical stimulation for living constructs realizing functionalization.
Combined with our research experiences in 3D bioprinting over years, this book is outlined in 14 chapters: commonly used 3D bioprinting methods are summarized first; then the design of bioink is put forward; several bioprinting approaches are elaborated afterward including coaxial bioprinting, digital light projection, direct ink writing, and liquid support bath‐assisted 3D printing; in the following parts, microgel‐based, microfiber‐based, and microfluidics‐based biofabrication approaches and their applications are meticulously illustrated; and a protocol of 3D bioprinting is well represented in the end to show several examples of complete bioprinting process.
We wish to thank the valuable support from everyone who contributed to this book. This book would never have been published without your effort. Thanks to Dr. Zeming Gu for help in writingChapter 1; thanks to Prof. Changxue Xu for help in writing Chapter 2; thanks to Mr. Peng Zhang for help in writing Chapter 3; thanks to Dr. Chaoqi Xie for help in writing Chapter 4; thanks to Dr. Yuan Sun for help in writing Chapter 5; thanks to Mr. Cheng Zhang for help in writing Chapter 6; thanks to Dr. Weijian Hua for help in writing Chapter 7; thanks to Dr. Mingjun Xie for help in writing Chapters 8, 9, and 14; thanks to Prof. Lei Shao for help in writing Chapters 10 and 11; thanks to Dr. Jing Nie for help in writing Chapters 12 and 13. Thanks to Zhengyi Zhang, Danyang Zhao, Lily Raymond, Heqi Xu, Matthew Warner, and Beatriz Godina for their help in completing this book. Thanks to Ms. Katherine Wong for editing the manuscript.
We sincerely hope that our readers will find the book professionally written, richly illustrated, accessible, and most importantly, intriguing. We would be flattered if this book attracts new researchers from different disciplines into the field of 3D bioprinting. Due to limited time and scholars with different backgrounds involved in compilation, there may be some unsatisfactory points in the writing style or content of this book. Therefore, readers are welcome to put forward criticism or suggestions for our further improvement.
4 May 2021
Yong He
Zhejiang University
Hangzhou, China
13D Bioprinting, A Powerful Tool for 3D Cells Assembling
1.1 What Is 3D Bioprinting?
3D printing, also known as additive manufacturing, is a layer‐by‐layer manufacturing approach, and it has been applied in many industrial applications and research fields. It could be thought of as an inverse process of potato cutting, assembling the chips or slices into integrity by certain rules. When 3D printing met biomedical engineering, 3D bioprinting was born. 3D bioprinting is an interdisciplinary science closely related to medicine biology, mechanical engineering, and material science. It can be divided into two concepts. Broadly speaking, 3D bioprinting refers to the use of 3D printing technology to achieve biomedical applications, such as the printing of medical aids, polymers, ceramics, or metal scaffolds [1–3]; in a narrow sense, this concept simply means 3D cells assembling through printing, therefore it can also be identified as cell printing or organ printing [4–6]. Here, this book is mainly focusing on the narrow viewpoint. A cartoon introduction of 3D bioprinting is illustrated in Figure 1.1.
In vitro bio‐manufacturing of tissues/organs has always been a great dream pursued by mankind, driven by two needs: organ transplantation and accurate tissue models. First, there is a huge shortage of organs for transplantation. In 2016, there were 160 000 organ transplant recipients, but only 16 000 organ donors in the United States [8]. The complexity of human organs is not only reflected in the mechanism of organ growth that has not been revealed by biology, but also in the reproduction of fine structure manufacturing. The use of 3D bioprinting technology to solve the shortage of organ transplants is far too optimistic at the present stage. Second, traditional methods utilizing 2D cell culture were applied for drug screening and medical mechanism studies. However, microenvironment in vivo is far more complex than the 2D cell culture, and in some cases, 2D models may lead to opposite results. 3D bioprinting technology can realize spatiotemporal directional manipulation of various cells and has become the most ideal method to construct a 3D cell‐laden structure in vitro.
In vitro models have undergone a meaningful revolution both in forms and functions: mini‐tissue, organ‐on‐a‐chip, and tissue/organ construct. Based on common bioprinting techniques, 3D mini‐tissue in forms of spheres, fibers, or other geometric shapes could be fabricated [9, 10]. These models contribute to the simulation of functional units with simple composition and independent operation, which can be applied in high‐throughput testing with a low dose. Besides, 3D bioprinting has been gradually involved in the setting up of organ‐on‐a‐chip devices because of its excellent customizability and cell compatibility [11]. Modified microfluidic systems could be constructed with biomaterials through 3D bioprinting, on which specified cells are loaded and routine reactions were carried out. And the interactions and cross‐talking between multiple organs can be well simulated by connecting different modules by means of microfluidic methods. Furthermore, 3D bioprinting has been further facilitated in the biofabrication of tissue/organ constructs with an inner channel network. A large number of 3D bioprinting strategies have been adopted in building 3D tissue/organ constructs with a vascular network, including coaxial bioprinting, projection‐based bioprinting, as well as the integration of 3D bioprinting and sacrificial templates.
Figure 1.1 Introduction of 3D bioprinting.
Source: He et al. [7]. Reproduced with permission of Springer Nature.
1.2 Evolution of 3D Bioprinting
As mentioned above, it is not practical to realize 3D bioprinting for full‐function organ transplantation at present. However, it is an undeniable fact that bioprinting techniques have come a long way. Decades ago, pioneers such as Vladimir Mironov, Gabor Forgacs, and Thomas Boland saw the natural combination of technologies including cell patterning and others, such as commercial inkjet printing, to build living structures that might one day be used for human organ transplantation [6, 12, 13]. A timeline for the evolution of bioprinting technology up to state‐of‐the‐art is illustrated in Table 1.1.
In 1984, Charles Hull invented stereolithography (SLA) for printing 3D objects from digital data, symbolizing the birth of 3D printing. Bioprinting was first demonstrated in 1988 while Klebe using a standard Hewlett–Packard (HP) inkjet printer to deposit cells by cytoscribing technology [14]. In 1996, Forgacs and coworkers drew a conclusion that apparent tissue surface tension was the macroscopic manifestation of molecular adhesion between cells and provided a quantitative measure for tissue cohesion [15]. In 1999, Odde and Renn first utilized laser‐assisted bioprinting to deposit living cells for developing analogs with complex anatomy [16]. In 2001, direct printing of a scaffold in the shape of a bladder and seeding of human cells took place [17]. In 2002, the first extrusion‐based bioprinting technology was reported by Landers et al., which was later commercialized as “3D‐Bioplotter” [18]. Wilson and Boland developed the first inkjet bioprinter in 2003 by modifying an HP standard inkjet printer [19]. Their team implemented cell‐loaded bioprinting with a commercial SLA printer a year after [20]. Also in 2004, 3D tissue with only cells (no scaffold) was developed. In 2006, electrohydrodynamic jetting was applied to deposit living cells [21]. Scaffold‐free vascular tissue was engineered through bioprinting by Norotte et al. in 2009 [22]. In 2012, in situ bioprinting was attempted by Skardal et al. on mouse models [23]. The following years saw the introduction of many new bioprinting products, such as articular cartilage and artificial liver in 2012, tissue integration with the circulatory system in 2014, and so on [24, 25]. In 2015, coaxial technology was adopted by Gao et al. for the fabrication of a tubular structure [4]. In 2016, Pyo et al. applied rapid continuous optical 3D printing based on digital light processing (DLP) [26]. In the same year, a cartilage model was manufactured by Anthony Atala's research group using an integrated tissue‐organ printer (ITOP) [27]. In 2019, Noor et al. succeeded in manufacturing a perfusable scale‐down heart [28], and a few months later, bioprinting of collagen human hearts at various scales based on the freeform reversible embedding of suspended hydrogels (FRESH) technology was achieved by Lee et al. [29].
Table 1.1 Timeline for bioprinting evolution.
Year
Development
1984
Stereo lithography was invented, representing the birth of 3D printing
1988
Bioprinting was first demonstrated by 2D micro‐positioning of cells
1996
Cells sticking together during embryonic development was observed
1999
First use of laser technology demonstrating 2D patterning of living cells
2001
3D printed synthetic scaffold for human ladder
2002
First extrusion‐based bioprinter was achieved
2003
First inkjet bioprinter was developed
2004
3D tissue with only cells (no scaffold) was presented
2009
Scaffold‐free vascular constructs were fabricated
2012
In situ bioprinting was realized on animals
2015
Tubular structure was printed by coaxial technology.
2016
Rapid continuous optical 3D printing based on projection (DLP) was applied
2016
Cartilage model was obtained by ITOP system
2019
Cardioid structure was first bioprinted
2019
Collagen human heart at various scales was built using FRESH technology
1.3 Brief Classification of 3D Bioprinting
Based on different printing principles, cell‐laden 3D bioprinting can be divided into three types: extrusion‐based, droplet‐based, and projection‐based bioprinting. Extrusion‐based bioprinting generates continuous fibers to set up the structures; droplet‐based bioprinting produces droplets as the basic unit for biofabrication and projection‐based bioprinting takes advantage of the properties of photosensitive materials by stacking 3D models layer‐by‐layer. Different approaches possess diverse characteristics aiming at various scenarios and have specific requirements for bioinks.
Extrusion‐based bioprinting is the most widely used method, which is suitable for a wide range of biocompatible materials. According to different liquid dispensing modes, pneumatic‐driven, piston‐driven, and screw‐driven extrusion systems are applied to extrude cell‐laden bioinks in the form of continuous filaments.
Droplet‐based bioprinting which employs discrete droplets stacked into constructs can be roughly divided into inkjet bioprinting [30], electrohydrodynamic jetting (EHDJ) [31], and laser‐assisted bioprinting (LAB) [32] based on different droplets forming principles. Thermal and piezoelectric‐driven technologies are most commonly used in inkjet bioprinting. EHDJ uses a high voltage motivated electric field to pull droplets out of the nozzle orifice. Changes in voltage certainly affect the size of each droplet, where the higher voltage leads to smaller droplets [33, 34]. LAB is a non‐contact, nozzle‐free bioprinting strategy used precisely to deposit bioink droplets. LAB technique includes laser‐guidance direct writing (LGDW) and laser‐induced forward transfer (LIFT). LGDW employs a light trap to guild cells onto a substrate, while LIFT uses a focused pulsed laser to induce partial evaporation of bioink coating to propel the biomaterial toward the receiving layer.
Projection‐based bioprinting solidifies light‐sensitive biomaterials to form constructs under precisely controlled lighting with high printing precision and fast printing speed. The most common use of projection‐based bioprinting is to print cell‐free scaffolds, where cells would be seeded post‐printing. Currently, however, cell‐laden projection‐based bioprinting has also been reported using DLP technology.
1.4 Evaluation of Bioinks
Generally speaking, 3D bioprinting has three steps: preparing bioinks, printing the soft live structures with multiple cells, and rebuilding the interaction among cells. And that is why developing appropriate bioinks has always been a significant part, as it affects every step that follows.
The performance of bioinks can be measured by three main factors: printability, biocompatibility, and mechanical property. Printability is to assess the formability of bioinks, where adjustable material viscosity, rapid transition from sol state to gel state, and a broad range of printing parameters are necessary. Biocompatibility is a measure of biomimicry that requires bioink and printed cells to be as similar as possible in the microenvironment in vivo. The mechanical property requires that the cured bioink be strong enough to hold subsequent culture and implantation. Perfusion and degradation might occur during bioprinted constructs culture in vitro, which requires considerable strength to support.
Therefore, the choice of bioink necessitates compromise among printability, biocompatibility, and mechanical property. Considering the requirements of the bioprinting process, cell growth and proliferation, and structural integrity, reasonable bioink design can be carried out according to the actual cell type and printing resolution requirements. But in fact, these three requirements of bioink are inherently contradictory in the mechanism. For example, the higher the viscosity of biological ink, the better the printability, and vice versa, the poorer the biocompatibility. Hence, bioink selection to meet the specific needs of different applications is a key step in bioprinting.
An ideal bioink would certainly be close to the natural extracellular matrix (ECM), and it would need to be adapted to match different types of cells. Therefore, it could not be better to add specific substances in bioinks that cells possibly need during proliferation and functionalization. For example, when bioprinting chondrocytes, the addition of HA, a common component of cartilage, can significantly promote later culture and functionalization.
Typical bioinks applied in bioprinting may include hydrogels, decellularized matrix components, microcarriers, tissue spheroids and strands, cell pellet, and/or some advanced bioinks such as multi‐material, interpenetrating network, nanocomposite, and supramolecular bioink, etc. [35, 36]. Among them, hydrogels are considered to be one of the most important biomaterials in bioinks, because of their outstanding capability of providing a viable microenvironment for cell adhesion, growth, and proliferation. Natural/synthetic hydrogels including alginate, fibrinogen, gelatin, collagen, silk fibroin, chitosan, agarose, pluronics, HA, GelMA, PEG, PEO, etc., have been found in countless applications in bioprinting. They are either ion‐sensitive, photosensitive, thermosensitive, enzyme‐sensitive, or pH‐responsive, so they can be easily gelated to form constructs before, during, and/or after bioprinting [37].
1.5 Outlook and Discussion
3D bioprinting technologies still need further improvement. The complexity of tissues and organs has brought great difficulties to accurate bioprinting. One of the major disadvantages of current bioprinting technologies is the low accuracy of bioprinting compared to natural tissues/organs. Most tissues/organs are more delicate than current bioprinting devices. Another common drawback of bioprinting is the slow speed of bioprinting of complex scale‐up structures, especially when it comes to multi‐material alternate biofabrication.
Vascularization is the basis of bioprinted structures. Same as the challenge of tissue engineering and regenerative medicine, ensuring adequate vascularization in bio‐manufactured structures is a key factor in 3D bioprinting. The effective construction of a multi‐scale perfusion vascular network and the promotion of its vascularization by mechanical or chemical stimulation are the basis of the biological fabrication of scale‐up constructs.
Functionalization is the primary goal for 3D bioprinting. Most of the current research is still focused on the manufacturing idea‐oriented printing process and mechanism, while functionalization is the core factor leading 3D bioprinting from basic research to practical application. In order to be functional, bioink needs to have excellent biocompatibility and mechanical properties to meet the requirements of nutrient perfusion and implantation. In addition, the construction of microenvironments that mimic in vivo scenarios, including mechanical and chemical stimuli such as perfusion culture and growth factors, is also critical for the functionalization of bioprinted structures.
Combined with the outlook of 3D bioprinting, there are several printing methods that are quite promising: DLP, coaxial bioprinting, and embedded bioprinting. Due to its intrinsic principle, DLP has a much higher printing resolution and speed than other bioprinting approaches. As a key application of 3D bioprinting technology, in vitro tissue models need to be standardized not only in sizes, but also in biological and mechanical properties, while DLP owns excellent uniformity and reproducibility compared to other methods. Additionally, coaxial bioprinting has become an increasingly popular extrusion‐based bioprinting method since it was introduced into the field of tissue engineering in 2015 [4], especially in the area of blood vessel biofabrication/vascularization. The biggest advantage of coaxial bioprinting is its ability to construct hierarchical tubular structures with tunable biological/mechanical properties. It is well known that hydrogels with good biocompatibility tend to have insufficient mechanical strength. Coaxial bioprinting can partly solve the problem with its core‐shell structure: core materials guarantee biocompatibility, while shell materials provide mechanical strength and vice versa. The use of sacrificial materials as the core material would also contribute to the convenient bioprinting of hollow tubular structures. Besides, embedded bioprinting allows anti‐gravity writing of 3D freeform constructs within yield stress and gel‐based supporting bath, which would be further removed post‐printing to retrieve models with desired shapes or channels. Other than traditional bioprinting approaches, it can achieve the fabrication of discrete patterns, which are not mechanically supported [38–40].
In addition to the challenges including bioinks design, bioprinting techniques, vascularization, and functionalization, issues such as cell sources, bioreactor construction, and even ethical problems also require considerable attention. 3D bioprinted fully clinical translation could take a long time until bio‐artificial tissues such as cartilage or skin, to be applied in transplantation. We all hope that 3D bioprinting can find its way from structural similarity into functional realization.
This book is organized into 14 chapters. This chapter “3D Bioprinting, A Powerful Tool for 3D Cells Assembling,” covers the definition, evolution, and classification of 3D bioprinting. Chapter 2 “Representative 3D Bioprinting Approaches” and Chapter 3 “Bioink Design” demonstrates a variety of commonly used 3D bioprinting methods in detail, and introduces the principle of bioink design. In Chapter 4 “Coaxial 3D Bioprinting,” Chapter 5 “Digital Light Projection‐Based 3D Bioprinting,” Chapter 6 “Direct Ink Writing for 3D Bioprinting Applications,” and Chapter 7 “Liquid Support Bath‐Assisted 3D Bioprinting,” four types of promising 3D bioprinting technologies and their applications are highlighted respectively. Chapter 8 “Bioprinting Approaches of Hydrogel Microgel,” and Chapter 9 “Biomedical Applications of Microgels” provides the manufacturing process and medical use of microgels. Chapter 10 “Microfiber‐Based Organoids Bioprinting for in vitro Model” and Chapter 11 “Large Scale Tissues Bioprinting” are mainly concerned with biofabricated organoids and scale‐up tissues. In Chapter 12 “3D Printing of Vascular Chips” and Chapter 13 “3D Printing of in vitro Models,” vascular chips and in vitro models by 3D printing approaches are well presented. Finally, Chapter 14 “Protocol of Typical 3D Bioprinting,” comes up with an integrated blueprint for 3D bioprinting.
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2Representative 3D Bioprinting Approaches
2.1 Introduction
Three‐dimensional (3D) bioprinting, defined as the spatial patterning of living cells, biomaterials, drugs, growth factors, and genes in a layer‐by‐layer manner, has been rapidly developed in recent years and widely used for fabricating living tissue and organ constructs for various biomedical applications [1–3]. It is envisioned as the first step toward the fabrication of functional replacement human organs in the future by bridging the gap between numerous biologics and integrated 3D living constructs.
In 3D bioprinting, building blocks are the fundamental units to construct living tissue and organ structures. Based on the methodology to form basic building blocks, current 3D bioprinting techniques can be divided into two categories: orifice‐based and orifice‐free [4]. In orifice‐based bioprinting, nozzles with microscale orifices are used as the tools to form either cell‐laden spheroids or strands as the building blocks. Inkjet bioprinting and extrusion bioprinting are two representative orifice‐based 3D bioprinting techniques. In orifice‐free bioprinting, different light sources are used as tools to form cellular layers instead of microscale orifices. Thus, the orifice‐free bioprinting is also known as light‐based bioprinting, as shown in Figure 2.1.
In orifice‐based bioprinting technologies, the typical biofabrication process consists of two main steps: printing and cross‐linking. The core function of the printing step is to form building blocks at a liquid state and then deposit them based on the designed trajectories. In inkjet bioprinting, different methods are used to form cell‐laden spheroids, namely droplets, as the building blocks, while in extrusion bioprinting, cell‐laden filaments are generated with continuous, cylindrical morphology to construct 3D structures. The main purpose of the subsequent cross‐linking step is to solidify the deposited droplets and/or filaments rapidly. Thus, the solidified droplets and/or filaments cannot only keep their shapes as designed, but also have the mechanical stiffness to support the following deposited droplets and/or filaments. By repeating the printing and cross‐linking steps, 3D structures can be fabricated by orifice‐based bioprinting technologies. As a result, classic orifice‐based bioprinting is performed in a “solidification‐while‐printing” fashion.
Orifice‐based 3D bioprinting technologies have many advantages. First, the independent cross‐linking step makes it possible to print biomaterials with different cross‐linking mechanisms. Thus, orifice‐based bioprinting technologies have a wider range of printable materials. Second, it is feasible to improve fabrication efficiency by simultaneously printing via numerous printheads with microscale nozzles. Third, orifice‐based bioprinting technologies provide a technical solution to print cellular constructs with different cell types. In both inkjet and extrusion bioprinting, multiple nozzles can be integrated within one printing system to deposit different cells through corresponding nozzles, facilitating the fabrication of spatially heterocellular constructs. Finally, current inkjet bioprinters and extrusion bioprinters can be easily implemented at affordable prices. This is due to the fact that ink‐jetting and extrusion are mature technologies and widely used in painting/printing and polymer/metal processing, respectively. However, orifice‐based 3D bioprinting technologies have complications. The main challenge during orifice‐based bioprinting is the high shear‐stress‐induced cell damage when cells flow through microscale nozzles. For inkjet bioprinting, the typical cell viability after printing is above 85% [1]. To form droplets with a well‐defined shape, cell density in inkjet bioprinting is always controlled at a low level, less than 106 cells/ml. For extrusion bioprinting, living cells are propelled through nozzles with microscale diameters to form filaments. During this process, high shear stress may bring damages to cell membranes and kill cells. Thus, typical cell viability in extrusion bioprinting falls in the range of 40–80%, even lower than that in inkjet bioprinting [1].
Figure 2.1 Classification of representative 3D bioprinting approaches.
Source: Yifei Jin.
Orifice‐free 3D bioprinting technologies cover several light‐based 3D printing approaches. Based on the different functionalities of a light source, they are divided into different subcategories. In matrix‐assisted pulsed‐laser evaporation (MAPLE) direct‐write, the laser beam is illuminated on biomaterial‐coated quartz to repel the localized materials away from the quartz in the morphology of droplets. This approach is also known as laser‐assisted 3D bioprinting. Since MAPLE uses droplets as the basic building blocks to construct 3D structures, the fabrication process is also composed of printing and cross‐linking steps, similar to that of inkjet bioprinting. However, different from inkjet bioprinting, living cells do not experience orifice‐induced high shear stress during droplet formation. As a result, cell viability in MAPLE is above 95%, much higher than those in both inkjet bioprinting and extrusion bioprinting. Despite the high cell viability, MAPLE is also constrained by some complications. On the one hand, preparation of coated quartz is time‐consuming, which makes the fabrication efficiency relatively low. On the other hand, the high price of laser system increases the cost of MAPLE significantly, which is another concern for clinical tissue engineering applications.
Aside from MAPLE, other light‐based 3D printing approaches combine the printing step with the cross‐linking step. Thus, cellular biomaterials are loaded in a container before printing and then cross‐linked in situ via different light sources. In particular, during stereolithography (SLA) and multi‐photon polymerization (MPP), one or multiple light spots are used to selectively cross‐link cellular biomaterials in a pixel‐by‐pixel or voxel‐by‐voxel manner, while during digital light projection (DLP) and computed axial lithography (CAL), each cross‐section of the designed 3D structure is projected via light pattern to cause the solidification of biomaterials in a layer‐by‐layer manner. Since no orifice is used during printing, the cell viability of these light‐based 3D printing approaches can be very high. In addition, the resolution of the printed 3D structures mainly depends on the size of the light spot and the accuracy of the light pattern, which can be easily controlled and improved. As a result, these 3D printing approaches are promising when creating 3D structures with high resolution. The disadvantage of the aforementioned light‐based 3D printing approaches is the limited material selection. To facilitate the printing process, biomaterials must be photocurable, which severely constrains the applications of these 3D printing approaches to fabricate living tissue and organ constructs from various non‐photocurable materials.
In the following sections, the mechanism, printable biomaterials, and representative biomedical applications of each 3D bioprinting strategy will be introduced in detail. In particular, Section 2.2 will focus on inkjet bioprinting technology. Section 2.3 will cover basic information on extrusion bioprinting, which will further be discussed in detail in Chapters 6 and 7. Section 2.4 will introduce some well‐developed light‐based 3D printing techniques such as MAPLE, SLA, MPP, and several newly proposed printing strategies, such as DLP and CAL.
2.2 Inkjet Bioprinting
Inkjet bioprinting is a droplet‐based 3D bioprinting technique in which cell‐laden droplets are used as the basic building blocks to construct complex 3D structures. Since this technique makes it feasible to accurately control the volumes and locations of the deposited bioink, it has been widely used in tissue engineering to fabricate spatially heterocellular constructs from various cellular inks. In this section, we will introduce this 3D bioprinting technique from three aspects: droplet formation mechanisms, materials, and representative biomedical applications.
2.2.1 Mechanisms of Droplet Formation
In inkjet bioprinting, a cellular bioink is squeezed through a microscale nozzle to generate cell‐laden droplets. By leveraging multiple factors including gravity, surface tension, and the fluid mechanics of the bioinks, the droplets are ejected from the nozzle and land on a receiving substrate. Based on different mechanisms to form droplets, inkjet bioprinting can be classified into three subcategories: (i) continuous‐inkjet (CIJ), (ii) drop‐on‐demand (DOD), and (iii) electrohydrodynamic (EHD) jet, as shown in Figure 2.2.
2.2.1.1 Continuous‐Inkjet Bioprinting
In CIJ bioprinting, cellular bioink is pushed through a nozzle under constant pressure to form a continuous liquid jet. Due to Rayleigh–Plateau instability, this liquid jet rapidly breaks up into a stream of droplets before landing on a substrate, as shown in Figure 2.2a. During the droplet formation, Rayleigh–Plateau instability plays an important role in which the liquid jet is perturbed by several factors including potential energy (gravity), surface energy (surface tension), and kinetic energy [5, 6]. When the wavelength of the perturbed jet is larger than the initial radius by a certain limit, the perturbation grows rapidly, breaking the jet into a series of droplets to minimize its potential energy. The condition to form droplets in CIJ can be simply modeled as a function of the number of waves per unit length (k) on the perturbed jet and the initial jet radius (R0). When kR0 is less than 1, the jet can distort itself into continuous droplets. The detailed information regarding Rayleigh–Plateau instability can be found in other published reports [7].
Figure 2.2 Mechanisms of droplet formation in inkjet bioprinting. (a) Continuous ink‐jetting based on Rayleigh–Plateau instability. (b) Drop‐on‐demand ink‐jetting including: (i) thermal ink‐jetting, (ii) piezoelectric ink‐jetting, and (iii) electrostatic ink‐jetting. (c) Electrohydrodynamic jetting based on an electric field.
Source: Yifei Jin.
2.2.1.2 Drop‐on‐Demand Inkjet Bioprinting
Different from CIJ, DOD inkjet bioprinting can generate droplets based on requirements. Thus, it is much easier to accurately control the locations of the deposited droplets and pattern cells and other biologics accordingly. As a result, DOD is the most popular inkjet bioprinting technique for cell bioprinting purposes. In DOD inkjet bioprinting, bioinks are transferred from fluid chambers to nozzles and squeezed out of the nozzles using different mechanisms. Since multiple nozzles can be used to print simultaneously, DOD inkjet bioprinting has the capacity of printing 3D cellular constructs with high efficiency. Currently, there are three main mechanisms to generate droplets during DOD ink‐jetting including: (a) thermal inkjet, (b) piezoelectric inkjet, and (c) electrostatic inkjet.
Thermal inkjet bioprinting
. In thermal inkjet bioprinting, a thermal actuator is integrated with a nozzle and heats bioink under a voltage pulse. This localized heating leads to the formation of a vapor bubble as shown in
Figure 2.2
b(i), which rapidly expands in the nozzle and generates pressure to the localized bioink, pushing it to the exit of the nozzle. Eventually, the bioink overcomes surface tension at the nozzle's exit and forms a droplet. Thermal inkjet bioprinting has been widely used to print living tissue constructs from various liquid bioinks such as
polyethylene glycol dimethacrylate
(
PEGDMA
), alginate, and cell suspensions. For example, Xu et al. [
8
] printed complex and heterogeneous 3D tissue constructs using a modified thermal inkjet printer. In their study, they mixed
human amniotic fluid‐derived stem cell
s (
hAFSC
s),
canine smooth muscle cell
s (
cSMC
s), and
bovine aortic endothelial cell
s (
bEC
s) separately with calcium chloride (CaCl
2
) as the cell‐laden inks and then printed them layer‐by‐layer to the predetermined locations in a sodium alginate‐collagen composite, as shown in
Figure 2.3
