Towards 4D Bioprinting

By Adrian Neagu

Towards 4D Printing presents the current state of three-dimensional (3D) bioprinting and its recent offspring, 4D bioprinting. These are attractive approaches to tissue engineering because they hold the promise of building bulky tissue constructs with incorporated vasculature. Starting with the discussion of 3D and 4D printing of inanimate objects, the book presents several 3D bioprinting techniques and points out the challenges imposed by living cells on the bioprinting process. It argues that, in order to fine-tune the bioprinter, one needs a quantitative analysis of the conditions experienced by cells during printing. Once the printing is over, the construct evolves according to mechanisms known from developmental biology. These are described in the book along with computer simulations that aim to predict the outcome of 3D bioprinting.In addition, the book provides the latest information on the principles and applications of 4D bioprinting, such as for medical devices and assistive technology. The last chapter discusses the perspectives of the field. This book provides an up-to date description of the theoretical tools developed for the optimization of 3D bioprinting, presents the morphogenetic mechanisms responsible for the post-printing evolution of the bioprinted construct and describing computational methods for simulating this evolution, and discusses the leap from 3D to 4D bioprinting in the light of the latest developments in the field. Most importantly, Towards 4D Printing explains the importance of theoretical modeling for the progress of 3D and 4D bioprinting.

• Presents theoretical tools needed for the optimization of the bioprinting process
• Describes the principles and implementation of computer simulations needed to predict the outcome of 3D bioprinting
• Analyzes the distinctive features of 4D bioprinting along with its applications and perspectives

• Language: English
• Publisher: Academic Press
• Release date: Nov 17, 2022
• ISBN: 9780128187456

Adrian Neagu

Dr. Adrian Neagu received his M.S. in Physics from the West University of Timisoara, Romania (1991). He worked at Freie Universität Berlin as a research fellow of the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD) (1992-1993). In 2002, he obtained his PhD in Statistical Physics from the Babes-Bolyai University of Cluj-Napoca, Romania. As a postdoctoral fellow in the research group led by Prof. Gabor Forgacs at the University of Missouri, Columbia, MO, USA, he studied the self-assembly of multicellular systems (2002-2003). He applied methods of statistical physics to develop computer simulations aimed at predicting the outcome of three-dimensional (3D) bioprinting of living tissue constructs [1-3]. He teaches Biophysics at the Victor Babes University of Medicine and Pharmacy Timisoara, as Associate Professor (2004-2006), and Professor (2006-present). In 2008, he was assigned Adjunct Professor at the University of Missouri, Columbia, USA. Here, as a visiting scholar, he worked on computational aspects of 3D tissue bioprinting as co-principal investigator of a National Science Foundation grant, FIBR-0526854 entitled "Understanding and employing multicellular self-assembly" (2006-2010). Dr. Neagu is coauthor of a patent on 3D tissue printing (United States Patent No. 8241905/14.08.2012), and editor of the Journal of 3D Printing in Medicine.

Book preview

Towards 4D Bioprinting

Adrian Neagu

Department of Biophysics, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania

Department of Physics, University of Missouri, Columbia, MO, United States

Table of Contents

Cover image

Title page

Copyright

Chapter 1. Introduction

1. Early milestones of 3D printing

2. Bioprinting—a form of biofabrication

Chapter 2. 4D printing: definition, smart materials, and applications

1. A technology inspired by life

2. Stimulus-responsive materials developed for 4D printing

3. Applications of 4D printing

Chapter 3. 3D and 4D printing of medical devices

1. From medical imaging to patient-matched anatomical models and surgical templates

2. The 3D printing of medical devices at the point of care

Chapter 4. 3D and 4D printing of assistive technology

1. Orthoses and prostheses

2. Assistive devices for daily living

Chapter 5. 3D Bioprinting techniques

1. Extrusion-based bioprinting

2. Droplet-based bioprinting

3. Light-based bioprinting

4. Spheroid-based bioprinting

Chapter 6. Theoretical methods for the optimization of 3D bioprinting: printability, formability, and cell survival

1. Theoretical tools in the optimization of extrusion-based bioprinting

2. Mathematical and computational methods for improving droplet-based bioprinting

3. Optimization of photopolymerization-based bioprinting

Chapter 7. Multicellular self-assembly

1. The differential adhesion hypothesis

2. Tissue surface tension

3. Tissue viscosity and the fusion of tissue spheroids

Chapter 8. Postprinting evolution of 3D-bioprinted tissue constructs

1. Monte Carlo models of single-cell resolution

2. Particle dynamics models

3. Phase field models

4. Lattice Boltzmann models

5. Conclusions and perspectives

Chapter 9. The definition of 4D bioprinting

1. Working definitions of 4D bioprinting

2. Potential refinements

Chapter 10. Applications of 4D bioprinting

1. Self-folding tubes

2. Shape morphing patches

Chapter 11. Perspectives of 3D and 4D bioprinting

1. Mathematical modeling

2. Emergent bioprinting techniques and materials

3. Potential applications

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1650, San Diego, CA 92101, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2023 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

ISBN: 978-0-12-818653-4

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Mara E. Conner

Acquisitions Editor: Carrie L. Bolger

Editorial Project Manager: Kyle Gravel

Production Project Manager: Fizza Fathima

Cover Designer: Christian J. Bilbow

Typeset by TNQ Technologies

Chapter 1: Introduction

Abstract

Besides a historical overview of 3D printing and bioprinting, this chapter provides the reader with the baggage needed to read the book and explore the cited references with minimal effort. First, the motivation of this book is explained and prerequisites are described. Then, the spotlight is put on the early days of 3D printing. Readers less passionate about the history of science and technology might still enjoy the story because it also pictures the landscape of ideas that sparked the invention of 3D printing. The historical account goes on by shifting the spotlight on the birth of bioprinting. Finally, the modern terminology of bioprinting and biofabrication is presented in the light of current literature.

Keywords

Bioengineering; Biological sciences; Computer graphics; Computer science; Dentistry; Drug delivery system; Drug development; Materials in biotechnology; Materials science engineering; Membrane system; Therapeutic procedure; Tissue; Tissue engineering

As fundamental sciences, engineering, and medicine join forces to provide personalized healthcare, we have the privilege to witness, and contribute to, the birth and growth of disruptive technologies rooted in three-dimensional (3D) printing. The emergence of affordable 3D printers compatible with a variety of 3D printable materials resulted in the widespread use of this technology, way beyond its original purpose of rapid prototyping.

Leveraging the ability of 3D printing to replicate seamless geometric complexity, anatomical models can be fabricated based on the patient's medical imaging. These can be used to plan the operation and discuss it with the patient. Also, patient-specific surgical guides and instruments can be manufactured on the spot to ensure a fast and effective intervention. Implantable medical devices can also be fabricated by 3D printing. In the case of pediatric patients, 4D-printed implants can change in shape and size to match the host's evolving anatomy. Also, people with disabilities can benefit from assistive devices tailored according to their needs using 3D and 4D printing.

Live tissue constructs can be manufactured from the patient's own cells by 3D bioprinting. They can serve as disease models and are useful for testing the efficacy of drugs. Nonetheless, the original motivation of cell printing research was to build organ replacements in the laboratory. Although twodecades of research did not bring us to that point, the biofabrication community is confident that, eventually, 3D-bioprinted implantable organs will alleviate donor organ shortage. Finally, 4D bioprinting will provide dynamical tissue constructs capable of recapitulating the response of certain organs to chemical and electrical cues, such as the vasoconstriction caused by caffeine or the motor activity of the gastrointestinal tract governed by nervous influxes.

The scientific literature dedicated to biomedical applications of 3D/4D printing and bioprinting is rapidly expanding. Besides sheer volume, the research output has also grown in diversity. Therefore, experienced investigators often struggle to keep up with the literature outside their own domain of expertise, and newcomers might be discouraged to enter the field. I wrote this book with the aim of providing a coherent overview of 3D/4D bioprinting terminology and technology. Also, I tried to showcase the rich spectrum of applications by selecting representative examples from the primary literature. I thank the authors for their great work and the publishers for the permission to reprint illustrations from the original papers. Since I prefer traveling to cooking, this book is very much like a tourist guide, as opposed to a collection of recipes. Actually, thinking of the discoveries responsible for the progress of bioprinting over the past twodecades, this book might be considered a treasure map. It provides background knowledge needed for understanding the original papers that shaped the current research landscape. Although the field will likely change between the time of writing and the date of publication, the foundations remain the same, and important milestones may inspire future discoveries.

Treating a vast field within a reasonable size limit required a careful choice of topics and depth of discussion. I apologize to those colleagues whose works are not mentioned in this book, as well as to those specialists who might find their specific topic of interest discussed a bit superficially. For deeper insight, the reader is invited to study the cited literature. With a bibliography of over 500 titles, the text might also serve as a reference guide for novice readers.

This book presents an interdisciplinary topic targeting a diverse audience. Therefore, I did my best trying to stick with straightforward language and avoid excessive jargon. I can only hope this did not result in scientific haiku (although I enjoy haiku, scientific or not).

To understand the text, the reader should only have basic knowledge of biology, chemistry, physics, and mathematics. Chapters are somewhat independent, but connections between them are often mentioned in the text. This book is intended for final-year undergraduate students, graduate students, and researchers willing to expand their horizons.

1. Early milestones of 3D printing

The central idea of 3D printing, that of building a bulky object one layer at a time, has been lingering around for about a century before Charles W. Hull filed his patent application for stereolithography on August 8, 1984 (Hull, 1986).

Actually, it did not just linger. The layer-by-layer fabrication of solid models was used extensively by morphologists since 1876, when the anatomist and embryologist Gustav Born, the father of Nobel laureate physicist Max Born (Born, 2002; Born et al., 1950), developed his wax plate method of embryo modeling (Born, 1876). He sectioned the embryos, using a microtome invented a few years earlier by Wilhelm His, and traced the enlarged histological sections onto wax plates. To preserve the aspect ratio of the anatomical structure, he magnified the cross-sectional image by the ratio of wax plate thickness to histological section thickness. Once the sections were drawn, he cut away the excess wax, preserving temporary support bridges to keep disconnected pieces together, and stacked successive layers on top of each other using vertical guides for precise alignment. His method was described in minute detail in a later paper (Born, 1883) and used since then (Gaunt & Gaunt, 1978; Hopwood, 1999).

Hull's patent was granted on March 11, 1986. It proposed to build a 3D object layer-by-layer by focusing an ultraviolet (UV) light beam onto the surface of a liquid photopolymer solution that turns solid when exposed to UV light. In his own words, stereolithography is a method and apparatus for making solid objects by successively ‘printing’ thin layers of a curable material, e.g., a UV curable material, one on top of the other (Hull, 1986).

By 1983, when this idea came to his mind, Charles (Chuck) Hull was working at a company that used UV light to coat furniture with tough plastic veneers (Hickey, 2014; Ponsford & Glass, 2014). Thus, he was familiar with UV curable materials, such as the acrylic-based Potting Compound 363 produced by Locktite Corporation. He was also aware of ongoing research on building 3D objects by selectively curing a fluid medium at prescribed points of intersection of multiple light beams (or one beam targeting sequentially the same point from different directions). In his patent application, Hull pointed out the drawbacks of those approaches—poor resolution and exposure control due to the interactions of electromagnetic waves with the media they cross on the way toward the target point. By contrast, the stereolithography apparatus (SLA) cured the surface layer of the fluid in millimeter-sized focal spots, which swiped the fluid surface under computer control. Once the first layer was cured, the print bed was lowered into the fluid-filled vat and fresh fluid covered the hardened layer. Selective light exposure brought about the hardening of the new layer, which adhered to the previous one (Hull, 1986).

In his initial setup, Chuck Hull employed a mercury short arc lamp whose output was focused onto a UV transmitting fiber optic bundle of 1mm in diameter. The bundle was fitted into the housing of a quartz lens that focused the UV beam onto the liquid surface, creating a light intensity of about 1W/cm² at the focal spot. To move the spot under computer control, the lens housing was attached to the pen carriage of a plotter. The patent application also mentions a UV laser as a potential light source and points out its major advantages: higher intensity and the feasibility of optical scanning. Remarkably, alternative setups of the SLA are also described (Hull, 1986), which are incorporated in modern-day digital light processing printers, as well as in the recently developed Fluid-supported Liquid Interface Polymerization (FLIP) 3D printing technique (Beh et al., 2021).

In the summer of 1984, 3weeks before Hull filed his patent application for stereolithography, three French scientists, Alain Le Méhauté, Olivier de Witte, and Jean Claude André filed theirs for a similar technique, in which laser light was delivered, via an optical fiber, into a photocurable liquid resin. The free end of the optical fiber was supposed to move, under computer control, within the bulk of the resin and cure nearby portions (Moussion, 2014). Their patent application, however, was abandoned by the Compagnie Générale d’Électricité (Le Méhauté's employer, which became Alcatel-Alsthom in 1991) and the Laser Consortium (CILAS) because their decision-makers did not see the commercial potential of the invention. I'm not bitter. I am proud of the innovative work we undertook and our efforts to promote technological innovation through the impetus of business and economic growth, declared Le Méhauté looking back at those days and the subsequent development of 3D printing. He also added that I have great respect for Hull who had the courage to initiate the creation of 3D Systems—a lesson of fair play and positive thinking (Mendoza, 2015).

The first 3D printing company in the world, 3D Systems, was cofounded by Chuck Hull in 1986 and remained at the forefront of 3D printing industry ever since. One year later, 3D Systems put the first 3D printer, SLA-1, on the market. With Chuck Hull as a Chief Technology Officer, 3D Systems continued to innovate and contributed substantially to the advancement of additive manufacturing (Our Story, n.d.).

Medical applications of 3D printing emerged soon thereafter (Mankovich et al., 1990). In their groundbreaking paper, Nicholas J. Mankovich, Andrew M. Cheeseman, and Noel G. Stoker from the University of California, Los Angeles, used an SLA printer to produce a physical model of a human skull starting from a computed tomography scan. They illustrate the power of the method, discuss the encountered difficulties (e.g., imaging artifacts), and propose a vast agenda for future research. They conclude that the usefulness of such models extends to surgical planning, radiation therapy, patient education, and physician education (see Chapter 3 to assess the accuracy of their insight).

In an interview given in 2014 to CNN, being asked what was most surprising to him in the evolution of 3D printing, Chuck Hull said To me, some of the medical applications. I didn't anticipate that, and as soon as I started working with some of the medical imaging people, it became pretty clear that this was going to work. But, you know, they told me, I didn't tell them (Ponsford & Glass, 2014).

2. Bioprinting—a form of biofabrication

2.1. The beginnings of bioprinting

The idea of using printers to build 3D tissue constructs emerged at the same time but independently of the SLA's commercialization. It sprang from repurposing inkjet printers and graphics plotters to create patterns of biomolecules on 2D substrates (Klebe, 1988).

Robert J. Klebe from the University of Texas Health Science Center, San Antonio, TX, printed fibronectin patterns on polystyrene films. To prevent cell attachment to fibronectin-free regions, the imprinted film was fixed on the bottom of a Petri dish with paraffin wax and treated for 10min with a 1% solution of thermally denatured bovine serum albumin dissolved in phosphate-buffered saline (PBS). Then, the film was washed twice with PBS, and cells were seeded on the film. This process has led to the formation of a 2D pattern of live cells anchored to the fibronectin layer. To build 3D tissue-like structures, Klebe prepared collagen gel sheets in molds made of a perforated sheet of graph paper sandwiched between two siliconized microscope slides. The collagen was seeded with cells and multiple layers could be glued together with additional collagen. Cell-type-specific extracellular matrix proteins and monoclonal antibodies were also proposed for positioning cells within individual layers, and stacking them was suggested as a method for building a 3D tissue construct. The take-home message of the paper was that further development of the proposed technology should aid in the production of artificial tissues which resemble natural tissues and organs (Klebe, 1988).

Cell sheet engineering became practical 2years later by coating cell culture dishes with a poly(N-isopropylacrylamide) (PNIPAAm) (Yamada et al., 1990). PNIPAAm is a thermoresponsive polymer, which is fully hydrated at room temperature, but at 32°C it becomes hydrophobic and, thereby, it enables cell attachment. (Indeed, cells cannot attach to highly hydrophilic surfaces because that would require displacing tightly bound water molecules.) Yamada and collaborators hypothesized that cells cultured in PNIPAAm-coated polystyrene dishes form confluent layers that can be detached by simply cooling the system below the polymer's transition temperature. This hypothesis proved to be correct and, despite the absence of micropatterning, cell sheet engineering produced clinically relevant applications by the turn of the century (Yang et al., 2007).

Biological cell printing was first accomplished by David J. Odde from the University of Minnesota, Minneapolis, MN, and Michael J. Renn from the Michigan Technological University, Houghton, MI (Odde & Renn, 1999). They used laser-induced optical forces to gently guide and deposit cells on a solid surface. They named their approach laser-guided direct writing (LGDW). The abilities of LGDW were illustrated by depositing chicken embryonic spinal cord cells onto a glass plate in a predefined arrangement. Cells were not harmed by the near-infrared laser beam that propelled them toward the substrate—deposited cells developed neurites, which indicates that they remained viable and functional. The range of LGDW could be extended to several centimeters by guiding the cells within hollow optical fibers. Fiber guiding was preferred over free guiding because (i) the fiber's lumen provided an unperturbed environment for cell movement and (ii) it enabled accurate positioning of cells by pointing the fiber's tip toward selected target points. Moreover, using multiple fibers would allow for printing a variety of cell types. Commenting on the potential applications of LGDW, Odde and Renn wrote that the ability to organize cells spatially into well-defined 3D arrays that closely mimic the native tissue architecture can potentially help in the fabrication of engineered tissue, and, based on their proof-of-concept experiments, they concluded that LGDW potentially allows the 3D patterning of cells using multiple cell types with cell placement at arbitrarily selected positions (Odde & Renn, 1999). The impatient reader might wish to take a look at Chapter 5 for further details on LGDW.

Along a different line of thinking, an important development originated from the work of Rüdiger Landers and Rolf Mülhaupt of the Albert-Ludwigs-Universität Freiburg, Germany (Landers & Mülhaupt, 2000). These authors constructed a 3D plotter capable of computer-controlled pneumatic extrusion of prepolymer solutions and pastes in liquid media to produce solid objects of complex shapes and intricate microarchitecture. They demonstrated the 3D plotting of silicone microdots and strands to build tubes and porous constructs akin to tissue engineering scaffolds. In their pioneering work, they noticed that 3D plotting does not involve harsh physicochemical factors and, therefore, is suitable to handle biomaterials loaded with live cells (Landers & Mülhaupt, 2000). The team also created agar hydrogel scaffolds by 3D plotting and made them appropriate for cell attachment via surface treatment—soaking in concentrated CaCl2 solution, rinsing with distilled water, and immersion in a diluted solution of hyaluronic acid and alginic acid (Landers et al., 2002). In a later work, dedicated to the fabrication of biodegradable polyurethane scaffolds, Rolf Mülhaupt's team proposed a new name for their technique, 3D bioplotting, even though their plotting material did not incorporate live cells, yet (Pfister et al., 2004). Rightfully so, since they pleaded for including cells and their works inspired the development of today's pneumatic extrusion-based bioprinters.

W. Cris Wilson, Jr., and Thomas Boland revolutionized the additive manufacturing of tissue constructs by adapting commercial inkjet printers to deliver cell suspensions (Wilson and Boland, 2003). This was the first time to use a jet-based instrument for fully automated, unattended printing of live cells. Wilson and Boland designed print heads comprising nine independently operated piezoelectric pumps. Each pump was fed with a cell suspension of its own via a flexible tubing and expelled droplets through a sterile needle. The pumps were controlled by a microchip programmable interface controller via an original software, whereas the print head movement remained under the control of the printer driver (rewritten to handle inks of different viscosities and electrical charges). Hewlett–Packard offered a generous gift to the tissue engineering community by providing the source code of the HP550C printer driver to Wilson and Boland. The modified printer was able to dispense 15nL droplets of bovine aortal endothelial cell suspension onto a Matrigel substrate, each droplet containing one or two cells. Postprinting cell viability was about 75%, remarkably high for a technology in its incipient phases. The authors suggested that cell damage might have been caused by dehydration since the minuscule droplets evaporated quickly. Hence the hydrogel substrate had a double role in protecting the dispensed cells: it cushioned their landing and kept them hydrated. In striking contrast with the bold title of the paper (the first to mention organ printing), its take-home message is careful and (with the hindsight of twodecades of progress) realistic: systematic three-dimensional cellular assemblies may become possible with the use of the ink-jet approach and these devices may have many potential applications, ranging from drug screening to tissue engineering (Wilson and Boland, 2003). The companion paper by Boland and coworkers, which appeared in the same issue of The Anatomical Record, provides further evidence that cell printing onto superimposed layers of thermosensitive gels has the potential to create 3D tissue constructs and explores avenues toward printing 3D organs. In particular, it suggests dispensing cell aggregates in successive layers contiguously to allow them to fuse on their own—a scenario that is quite common in developmental biology (Boland et al., 2003). For a deeper insight into using cell aggregates as building blocks of 3D-printed tissues, the team contacted Gabor Forgacs from the University of Missouri–Columbia, MO, who had spent more than a decade investigating the biomechanical properties of cell aggregates and also had a solid experience in developmental biology (Forgacs & Newman, 2005). Their joint paper became the most cited work on organ printing (Mironov et al., 2003).

Today, a search for the term organ printing returns over 140,000 hits on Google. Back then, the terminology was so new that even the researchers felt the urge to explore the thin borderline between science and fiction (Jakab et al., 2004). The idea of printing an implantable organ was in utter contrast with the possibilities of the hardware and materials within reach. Indeed, organ printing is still a long-term goal (Ng et al., 2019), but it seems more tangible today, as demonstrated by many examples presented in the forthcoming chapters. As the field matured, its terminology became more nuanced: 3D bioprinting became the popular term for the additive manufacturing of live structures, whereas the cell-containing materials dispensed by 3D bioprinters are called bioinks. Bioprinting got embedded into the broader field of biofabrication, which encompasses a vast set of bottom-up technologies of tissue engineering and regenerative medicine. Novel 3D bioprinting techniques mushroomed during the past decade. Chapter 5 presents the most common ones in detail, Chapter 11 discusses recent advances, and new ones will likely emerge by the time this book reaches its audience.

The next section is dedicated to the definitions of the terms bioprinting and biofabrication—discussed in 8 million and 11 million Google documents, respectively.

2.2. The terminology of biofabrication

The term bioprinting was coined at The First International Workshop on Bioprinting and Biopatterning, held in September 2004, in Manchester, UK, organized by Brian Derby from the University of Manchester; Douglas B. Chrisey from the U.S. Naval Research Laboratory, Washington, DC, USA; Richard K. Everett from ONR Global, London, UK; and Nuno Reis from Universidade da Beira Interior, Covilhã, Portugal. At this seminal workshop, bioprinting was defined as the use of material transfer processes for patterning and assembling biologically relevant materials – molecules, cells, tissues, and biodegradable biomaterials – with a prescribed organization to accomplish one or more biological functions (Mironov et al., 2006). Technical challenges faced by the new technology were recognized, such as the need for a fluid vehicle that shortly after printing requires consolidation and should consequently behave as a viscoelastic solid, and this phase change must occur without damage to the biochemicals, cells, or more complex units within the fluid, establishing important guidance for material development. Despite many uncertainties about the future of the new technology, the participants decided to organize periodic meetings and pondered the opportunity of initiating a professional society and, perhaps, a journal (Mironov et al., 2006).

A dedicated journal, entitled Biofabrication, was introduced at the fourth meeting, organized in July 2009, in Bordeaux, France. Published by IOP Science, Biofabrication grew rapidly in content and prestige. In the meantime, also other high-quality journals emerged, including Bioprinting (by Elsevier) and International Journal of Bioprinting (by WHIOCE Publishing). The Bordeaux meeting has also provided an updated definition of bioprinting: the use of computer-aided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies (Guillemot et al., 2010).

In the first issue of the new journal, the term biofabrication was defined as the production of complex living and nonliving biological products from raw materials such as living cells, molecules, extracellular matrices, and biomaterials (Mironov et al., 2009). This definition pertains to the field of tissue engineering and regenerative medicine because, since 1994, the term biofabrication has been used in several disciplines, with a variety of meanings (Groll et al., 2016).

The fifth meeting, in October 2010, Philadelphia, USA, marked an important milestone: the International Society for Biofabrication (ISBF) was founded (International Society for Biofabrication (ISBF), 2010). Nowadays, the annual meetings organized by the ISBF, the so-called International Conferences on Biofabrication, attract hundreds of participants from all over the world.

The growing popularity and research production, however, often lead to inconsistent terminology. To fight this tendency, prominent members of the ISBF periodically publish perspective papers to clarify the conceptual framework of this vivid research field (Groll et al., 2019, 2016; Moroni et al., 2018). In particular, the definition of the field itself has been revisited to accommodate novel approaches. Currently, biofabrication is achieved via two main strategies: bioprinting and bioassembly. The latter is defined as the fabrication of hierarchical constructs with a prescribed 2D or 3D organization through automated assembly of pre-formed cell-containing fabrication units generated via cell-driven self-organization or through preparation of hybrid cell-material building blocks, typically by applying enabling technologies, including microfabricated molds or microfluidics. For bioassembly, one starts from preformed multicellular units such as organoids, cell sheets, or cell fibers. Composed of cells and their extracellular matrix, such units can be obtained through multicellular self-organization in specific environments created by microfluidics and micromolding techniques. A variety of automated methods can be applied to achieve bioassembly depending on the size and geometry of the building blocks (e.g., in the case of cell fibers, one can use methods of textile industry, such as weaving, winding, and knitting). Thus, in the context of tissue engineering and regenerative medicine, according to the currently accepted working definition, biofabrication consists in the automated generation of biologically functional products with structural organization from living cells, bioactive molecules, biomaterials, cell aggregates such as micro-tissues, or hybrid cell-material constructs, through bioprinting or bioassembly and subsequent tissue maturation processes (Groll et al., 2016). To assist newcomers and consolidate the professional jargon of active investigators, a vast review article has been published by biofabrication experts, which includes a glossary of terms and a description of the major technologies used in biofabrication (Moroni et al., 2018).

Together with its many (but carefully selected) references, this book is meant to help readers eager to explore the rapidly changing world of bioprinting.

References

1. Beh C.W, Yew D.S, Chai R.J, Chin S.Y, Seow Y, Hoon S.S. A fluid-supported 3D hydrogel bioprinting method.  Biomaterials . 2021;276:121034. doi: 10.1016/j.biomaterials.2021.121034.

2. Boland T, Mironov V, Gutowska A, Roth E.A, Markwald R.R. Cell and organ printing 2: Fusion of cell aggregates in three-dimensional gels.  Anatomical Record Part A Discoveries in Molecular Cellular and Evolutionary Biology . 2003;272:497–502. doi: 10.1002/ar.a.10059.

3. Born G. Ueber die Nasenhöhlen und den Thränennasengang der Amphibien.  Morphologisches Jahrbuch . 1876;2:577–646.

4. Born G. Die plattenmodellirmethode.  Archiv für Mikroskopische Anatomie . 1883;22:584–599.

5. Born G.V.R. The wide-ranging family history of Max Born.  Notes and Records of the Royal Society of London . 2002;56:219–262. http://www.jstor.org/stable/3557669.

6. Born M, Brandt W, Born G. In memoriam Gustav Born, experimental embryologist.  Acta Anatomica . 1950;10:466–475. doi: 10.1159/000140488.

7. Forgacs G, Newman S.A. Biological physics of the developing embryo. Cambridge University Press; 2005. https://books.google.ro/books?id=rUyVWQhk7CkC.

8. Gaunt W.A, Gaunt P.N.  Three dimensional reconstruction in biology . University Park Press; 1978.

9. Groll J, Boland T, Blunk T, Burdick J.A, Cho D.-W, Dalton P.D, Derby B, Forgacs G, Li Q, Mironov V.A, Moroni L, Nakamura M, Shu W, Takeuchi S, Vozzi G, Woodfield T.B.F, Xu T, Yoo J.J, Malda J.Biofabrication: Reappraising the definition of an evolving field.  Biofabrication . 2016;8:013001. doi: 10.1088/1758-5090/8/1/013001.

10. Groll J, Burdick J.A, Cho D.W, Derby B, Gelinsky M, Heilshorn S.C, Jüngst T, Malda J, Mironov V.A, Nakayama K, Ovsianikov A, Sun W, Takeuchi S, Yoo J.J, Woodfield T.B.F.A definition of bioinks and their distinction from biomaterial inks.  Biofabrication . 2019;11:013001. doi: 10.1088/1758-5090/aaec52.

11. Guillemot F, Mironov V, Nakamura M. Bioprinting is coming of age: Report from the international conference on bioprinting and biofabrication in Bordeaux (3B’09).  Biofabrication . 2010;2:010201. doi: 10.1088/1758-5082/2/1/010201.

12. Hickey S. Chuck Hull: The father of 3D printing who shaped technology. The Guardian; June 22, 2014. www.theguardian.com/business/2014/jun/22/chuck-hull-father-3d-printing-shaped-technology.

13. Hopwood N. Giving body to embryos. Modeling, mechanism, and the microtome in late nineteenth-century anatomy.  Isis . 1999;90:462–496. doi: 10.1086/384412. .

14. Hull C.W. Apparatus for production of three-dimensional objects by stereolithography.  United States Patent 4,575,330 . March 11, 1986. https://patentimages.storage.googleapis.com/5c/a0/27/e49642dab99cf6/US4575330.pdf.

15. International Society for Biofabrication (ISBF). https://biofabricationsociety.org/about/, 2010.

16. Jakab K, Neagu A, Mironov V, Forgacs G. Organ printing: Fiction or science.  Biorheology . 2004;41:371–375.

17. Klebe R.J. Cytoscribing: A method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues.  Experimental Cell Research . 1988;179:362–373. doi: 10.1016/0014-4827(88)90275-3.

18. Landers R, Hübner U, Schmelzeisen R, Mülhaupt R. Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering.  Biomaterials . 2002;23:4437–4447. doi: 10.1016/S0142-9612(02)00139-4.

19. Landers R, Mülhaupt R. Desktop manufacturing of complex objects, prototypes and biomedical scaffolds by means of computer-assisted design combined with computer-guided 3D plotting of polymers and reactive oligomers.  Macromolecular Materials and Engineering . 2000;282:17–21. doi: 10.1002/1439-2054(20001001)282:1<17::AID-MAME17>3.0.CO;2-8.

20. Mankovich N.J, Cheeseman A.M, Stoker N.G. The display of three-dimensional anatomy with stereolithographic models.  Journal of Digital Imaging . 1990;3:200. doi: 10.1007/BF03167610.

21. Mendoza H.R. Alain Le Méhauté, the man who submitted patent for SLA 3D printing before Chuck Hull. May 15, 2015. https://3dprint.com/65466/reflections-alain-le-mehaute/.

22. Mironov V, Boland T, Trusk T, Forgacs G, Markwald R.R. Organ printing: Computer-aided jet-based 3D tissue engineering.  Trends in Biotechnology . 2003;21:157–161. http://www.sciencedirect.com/science/article/pii/S0167779903000337.

23. Mironov V, Reis N, Derby B. Review: Bioprinting: A beginning.  Tissue Engineering . 2006;12:631–634. doi: 10.1089/ten.2006.12.631.

24. Mironov V, Trusk T, Kasyanov V, Little S, Swaja R, Markwald R. Biofabrication: A 21st century manufacturing paradigm.  Biofabrication . 2009;1:022001. doi: 10.1088/1758-5082/1/2/022001.

25. Moroni L, Boland T, Burdick J.A, De Maria C, Derby B, Forgacs G, Groll J, Li Q, Malda J, Mironov V.A, Mota C, Nakamura M, Shu W, Takeuchi S, Woodfield T.B.F, Xu T, Yoo J.J, Vozzi G.Biofabrication: A guide to technology and terminology.  Trends in Biotechnology . 2018;36:384–402. doi: 10.1016/j.tibtech.2017.10.015.

26. Moussion A. Rencontre avec Alain Le Méhauté, l’un des pères de l’impression 3D! (Meeting Alain Le Méhauté, one of the fathers of 3D printing!. September 17, 2014. www.primante3d.com/inventeur/.

27. Ng W.L, Chua C.K, Shen Y.-F. Print me an organ! Why we are not there yet.  Progress in Polymer Science . 2019;97:101145. doi: 10.1016/j.progpolymsci.2019.101145.

28. Odde D.J, Renn M.J. Laser-guided direct writing for applications in biotechnology.  Trends in Biotechnology . 1999;17:385–389. doi: 10.1016/S0167-7799(99)01355-4.

29. Our Story. (n.d.). 3D systems. Retrieved from www.3dsystems.com/our-story.

30. Pfister A, Landers R, Laib A, Hübner U, Schmelzeisen R, Mülhaupt R. Biofunctional rapid prototyping for tissue-engineering applications: 3D bioplotting versus 3D printing.  Journal of Polymer Science Part A: Polymer Chemistry . 2004;42:624–638. doi: 10.1002/pola.10807. .

31. Ponsford M, Glass N. The night I invented 3D printing. CNN; February 14, 2014. https://edition.cnn.com/2014/02/13/tech/innovation/the-night-i-invented-3d-printing-chuck-hall/index.html.

32. Wilson Jr. W.C, Boland T. Cell and organ printing 1: Protein and cell printers.  The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology . 2003;272A:491–496. doi: 10.1002/ar.a.10057.

33. Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells.  Makromolekulare Chemie, Rapid Communications . 1990;11:571–576. doi: 10.1002/marc.1990.030111109.

34. Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M, Ohki T, Nishida K, Okano T.Reconstruction of functional tissues with cell sheet engineering.  Biomaterials . 2007;28:5033–5043. doi: 10.1016/j.biomaterials.2007.07.052.

Chapter 2: 4D printing: definition, smart materials, and applications

Abstract

Four-dimensional (4D) printing is a relatively new concept, less than a decade old. This chapter provides a brief history of 4D printing and locates it in the broader research field of programmable matter. Then, 4D printing is defined and stimulus-responsive (smart) materials are presented, which enable the leap from 3D to 4D printing: an anticipated response of the printout as a result of a predetermined stimulus, such as a change in temperature or water immersion. Finally, the discussion turns to biomedical applications of 4D printing. Specific examples of endoluminal devices, soft actuators, soft swimmers, and tissue-like assemblies of aqueous droplets in oil demonstrate that 4D printing is a powerful technology for creating smart objects to be used in health care.

Keywords

Bioengineering; Biotechnology; Materials characterization; Materials class; Materials physics; Materials processing; Materials property; Mechanics; Soft matter physics

The term 4D printing has been coined by Skylar Tibbits