Additive Manufacturing of Biopolymers: Handbook of Materials, Techniques, and Applications 9780323951517

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Additive Manufacturing of

BIOPOLYMERS

Handbook of Materials, Techniques, and Applications

Edited by

MEHRSHAD MEHRPOUYA HENRI VAHABI

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States 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: 978-0-323-95151-7 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Candice Janco Acquisitions Editor: Anita Koch Editorial Project Manager: Kyle Gravel Production Project Manager: Bharatwaj Varatharajan Cover Designer: Miles Hitchen and Jeroen van Kekem Typeset by TNQ Technologies

Contents 1. Additive manufacturing of biopolymers 2. Additive manufacturing and 3D printing techniques for biopolymers 3. Biopolymers in additivemanufacturing 4. 3D printing of biopolymer-based hydrogels 5. 3D printing of fire-retardant biopolymers 6. 3D printing of biopolymer composites and nanocomposites 7. 3D printing of shape-switching biopolymers 8. 4D printing of biopolymers 9. Post-processing methods for 3D printed biopolymers 10. 3D printed bio-based polymers and hydrogels for tissue engineering 11. 3D printed biopolymers for medical applications and devices 12. Potential applications of 3D and 4D printing of biopolymers 13. 3D printing with biopolymers: toward a circular economy Index

Foreword

It is important that we have books like this one, particularly in times like we have now, with major concerns over the polluting effects of plastics. It is interesting to note that the first synthetic polymers were invented to overcome the environmental concerns around the demand for ivory for the making of the likes of billiard balls. If we only knew then what we do now. I’m sure there was not even an inkling in the minds of the first inventors of synthetic polymers that this would be the start of one of the largest industrial sectors the world has ever seen. But now, we are faced with huge dilemmas over how we sustain an industry without causing further and lasting damage to our planet. Throughout my academic career and at close quarters, I have watched additive manufacturing grow from humble beginnings as a means to rapidly create product prototypes into what is now a major manufacturing technology. We will undoubtedly see it develop even further as it becomes accepted in more domains and as a more widespread, cost-effective solution. We must therefore be careful about how we develop the future of this technology, particularly in terms of how we generate the feedstock materials for an ever-increasing number of additive manufacturing machines. In short, we should be developing more biopolymers. This book is therefore not only important for the useful solutions and advice it provides to support the development of biopolymers, but also it acts as a beacon, providing the direction for an ethical and sustainable future. Ian Gibson Professor of Design Engineering Department of Design, Production, and Management Director of Fraunhofer Innovation Platform for Advanced Manufacturing University of Twente Enschede, The Netherlands

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Contributors

Mohsen Akbari Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada; Centre for Advanced Materials and Related Technologies, University of Victoria, Victoria, BC, Canada; Terasaki Institute for Biomedical Innovation, Los Angeles, CA, United States; School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada; Biotechnology Center, Silesian University of Technology, Gliwice, Poland Jia An Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore Esfandyar Askari Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada Marcos Batistella Polymeres Composites et Hybrides (PCH), IMT Mines Ales, Ales, France Mahdi Bodaghi Department of Engineering, School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom Pilar Bolumburu Materiom, London, United Kingdom Declan Devine The Polymer, Recycling, Industrial, Sustainability and Manufacturing Research Institute, Technological University of the Shannon: Midlands Midwest, Athlone, Co. Westmeath, Ireland Elizabeth Diederichs Waterloo Composite Biomaterial Systems Laboratory, University of Waterloo, Waterloo, ON, Canada Lisa Elviri Food and Drug Department, University of Parma, Parma, Italy Rudy Folkersma Professorship Sustainable Polymers, NHL Stenden University of Applied Sciences, Emmen, the Netherlands Alysia Garmulewicz Department of Management, Faculty of Management and Economics, University of Santiago of Chile, Santiago, Chile; CABDyN Complexity Centre, University of Oxford, Oxford, United Kingdom; Materiom, London, United Kingdom Sanaz S. Hashemi Waterloo Composite Biomaterial Systems Laboratory, University of Waterloo, Waterloo, ON, Canada

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Contributors

Andrew Healy Department of Pharmaceutical Sciences and Biotechnology, Technological University of the Shannon: Midlands Midwest, Athlone, Co. Westmeath, Ireland Wei Min Huang School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore Gavin Keane The Polymer, Recycling, Industrial, Sustainability and Manufacturing Research Institute, Technological University of the Shannon: Midlands Midwest, Athlone, Co. Westmeath, Ireland Ana C. Lemos de Morais Professorship Sustainable Polymers, NHL Stenden University of Applied Sciences, Emmen, the Netherlands; Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands Kah Fai Leong Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore Katja Loos Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands Jose-Marie Lopez-Cuesta Polymeres Composites et Hybrides (PCH), IMT Mines Ales, Ales, France Mehrshad Mehrpouya Faculty of Engineering Technology, University of Twente, Enschede, the Netherlands Dibakar Mondal Waterloo Composite Biomaterial Systems Laboratory, University of Waterloo, Waterloo, ON, Canada Wei Long Ng HP-NTU Digital Manufacturing Corporate Lab, Singapore; Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University (NTU), Singapore Falguni Pati Biofab Lab, Department of Biomedical Engineering, IIT Hyderabad, Sangareddy, Telangana, India Haresh Patil Waterloo Composite Biomaterial Systems Laboratory, University of Waterloo, Waterloo, ON, Canada Damien Rasselet Polymeres Composites et Hybrides (PCH), IMT Mines Ales, Ales, France Giulia Remaggi Food and Drug Department, University of Parma, Parma, Italy Charlene Smith Materiom, London, United Kingdom

Contributors

Filippos Tourlomousis National Centre for Scientific Research Demokritos, Attica, Greece; Superlabs AMKE, Marousi, Greece; Biological Lattice Industries Corp, Boston, MA, United States Henri Vahabi Universite de Lorraine, CentraleSupelec, LMOPS, Metz, France Vincent S.D. Voet Professorship Sustainable Polymers, NHL Stenden University of Applied Sciences, Emmen, the Netherlands Thomas L. Willett Waterloo Composite Biomaterial Systems Laboratory, University of Waterloo, Waterloo, ON, Canada Fengwei Xie School of Engineering, Newcastle University, Newcastle upon Tyne, United Kingdom Wai Yee Yeong HP-NTU Digital Manufacturing Corporate Lab, Singapore; Singapore Centre for 3D Printing (SC3DP), School of Mechanical and Aerospace Engineering, Nanyang Technological University (NTU), Singapore Alessandro Zaccarelli Food and Drug Department, University of Parma, Parma, Italy Lubna Zeenat School of Engineering, Deakin University, Geelong, VIC, Australia; Biofab Lab, Department of Biomedical Engineering, IIT Hyderabad, Sangareddy, Telangana, India Ali Zolfagharian School of Engineering, Deakin University, Geelong, VIC, Australia

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CHAPTER 1

Additive manufacturing of biopolymers Mehrshad Mehrpouyaa and Henri Vahabib

a Faculty of Engineering Technology, University of Twente, Enschede, the Netherlands; bUniversit
e de Lorraine, CentraleSup
elec, LMOPS, Metz, France

1. Introduction Additive manufacturing (AM), also called 3D printing, is a computer-controlled process that can create 3D objects by depositing the material layer-by-layer (1). This technology emerged a few decades ago and has grown rapidly because of providing a unique capability in the fabrication of complex shapes and highly customizable products with minimum waste (2). This makes AM a better candidate compared to conventional manufacturing methods, which are often subtractive and waste a lot of energy and materials. Currently, AM technologies are the key components of the last industrial revolution, namely industry 4.0, since they can significantly decrease manufacturing steps, time, and material waste (3,4). Particularly, they offer the possibility to apply geometrical design for specific needs and have already many applications in a variety of industries such as aerospace, construction, biomedical, and automotive. In recent decades, there have been many concerns about the negative impacts of environmental pollution from fossil fuels and waste from petrochemical products. Although many materials have been developed for various AM techniques, selecting processable, environmentally safe, and printer-friendly materials based on fabrication and performance requirements remains a challenge. Much research has gone into finding alternatives to petroleum-based products that are renewable and biodegradable, posing less harm to the environment (5). Biopolymers are one such possible solution to the problem because they are, in some cases, biodegradable materials obtained from renewable raw materials (6,7). As one might expect, there are challenges related to biopolymers such as their limited rate of production, cost of production, and their performance.

2. Biopolymers Biopolymer is a general term used for all polymers that are biobased and/or biodegradable. Besides, biodegradability means that microorganisms are able to degrade the material into other substances such as water, carbon dioxide, etc. It is possible to have biobased/nonbiodegradable biopolymers and also to have fossil fuel-based/biodegradable biopolymers.

Additive Manufacturing of Biopolymers ISBN 978-0-323-95151-7, https://doi.org/10.1016/B978-0-323-95151-7.00002-8

© 2023 Elsevier Inc. All rights reserved.

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For example, poly(caprolactone) (PCL) recognized as a biopolymer is obtained from nonrenewable resources (petroleum-based) and biodegradable, and biobased poly(ethylene terephthalate) (bio-PET) is not biodegradable but obtained from renewable resources. It is worth mentioning that the term “bioplastic” is a general term usually used instead of biopolymer, and against non-biobased, non-biodegradable (non-compostable) conventional polymers obtained from fossil sources (e.g., crude oil or natural gas). Also, the “bioplastic” term is not generally used in the case of biobased “natural” biopolymers such as collagen or cellulose. Fig. 1.1 shows the classification of biopolymers from this point of view. Biopolymer sources are numerous and can be obtained from five categories including animals, plants, microorganisms, chemical synthesis of natural origin, and chemical synthesis of fossil sources. Therefore, biopolymers can also be classified based on their source (natural, synthetic, renewable, fossil), biodegradability, applications, and process technologies (see for example Chapters 3 and 11). Here, we present a classification based on the origin of biopolymers which is divided into three families: (a) biopolymers obtained directly from natural resources, such as polysaccharides proteins, and lipids; (b) obtained by chemical synthesis from biobased monomers, such as polyhydroxyalkanoates (PHA) and poly(lactic acid) (PLA); (c) produced from fossil resources, such as PCL and polybutylene adipate terephthalate (PBAT). Fig. 1.2 illustrates this classification in more detail including the sub-groups and some examples for each. The production of biopolymers currently remains low (2.42 million tons/year) compared with conventional polymers (335 million tons/year) (8). However, forecasts show continuous growth in the production of biopolymers to reach 7.6 million tons/

Fig. 1.1 General classification of biopolymers in terms of “source and biodegradability.”

Additive manufacturing of biopolymers

Fig. 1.2 Classification of biopolymers and some examples. PHAs, polyhydroxyalkanoates; PLA, poly(lactic acid); PGA, poly(glycolic acid); PCL, polycaprolactone; PBAT, polybutylene adipate terephthalate.

year in 2026. Fig. 1.3 displays the global current production capacities of different biopolymers. It can be observed that PBAT, PLA, polyethylene (PE), and starch-based biopolymers are among the most produced biopolymers. As shown in Fig. 1.4, biopolymers are currently used in several applications including the agriculture, pharmaceutical, packaging, food, and cosmetics industries as well as biomedicine and biomedical, thanks to their unique properties (see Chapters 10, 11, and 12). It is notable that the packaging sector represents the biggest market segment of biopolymers, accounting for 48% of the total market (around 1.15 million tons) (10).

3. Biopolymers in additive manufacturing The use of biopolymers in additive manufacturing can be considered from several points of view. Nowadays, the negative impact of conventional petroleum-based polymers on the environment is at the center of attention. As a result, the initial incentive is tied to the development of sustainable and environmentally friendly materials (11). These negative effects are related to the accumulation of a massive amount of plastic waste in water and soil. Microplastics impact microorganisms and disrupt marine ecology (12,13). Also, the huge amount of polymer waste in nature is a source of pollution of soil and is harmful not

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Fig. 1.3 Global world production capacities of biopolymers by material type. PE, polyethylene; PET, poly(ethylene terephthalate); PA, polyamide; PP, polypropylene; PEF, polyethylene furanoate; PTT, polytrimethylene terephthalate; PBAT, polybutylene adipate terephthalate; PBS, polybutylene succinate; PLA, poly(lactic acid); PHA, polyhydroxyalkanoate. (Data obtained from (8).)

Fig. 1.4 Applications of biopolymers including pharmacy, medicine, biomedical, packaging, food, cosmetic, and agriculture. (Modified from (9).)

Additive manufacturing of biopolymers

only to soil animals but also to human health. At this moment, this is a significant hazard to the ecological system, particularly water resources and the oceans. This is because they have a direct impact on the food chain and consequently on human health. Moreover, these polymer wastes contain different kinds of additives or their degradation products, such as pigments, plasticizers, halogenated flame retardants, etc., that contaminate soil and water resources (14,15). Also, the limitations of access to petroleum-based resources are also one of the motivations for developing biobased polymers (16). Furthermore, it could be stressed that the development of biobased polymers allows the supply of polymer material to be enhanced and diversified. Hence, new opportunities are emerging for big companies in the agrochemical area. In this regard, the circular economy and sustainability need to be considered in the development of biopolymers for additive manufacturing (see Chapter 13). So far, a wide range of biopolymers has been developed for use in various types of additive manufacturing technologies. However, there are still many ongoing research projects to introduce innovative biopolymers with novel features and functional performance as well as enhanced biodegradability, biocompatibility, etc.

4. Classification of AM techniques for biopolymers In recent years, various AM techniques have been developed for the fabrication of biopolymers. This section summarizes the most common AM techniques used for biopolymers. As shown in Fig. 1.5, AM techniques for biopolymers are categorized into six groups including vat polymerization, material jetting, materials extrusion, binder jetting, powder bed fusion, sheet lamination.

Fig. 1.5 Classification of main techniques for additive manufacturing of biopolymers.

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Each of these AM techniques uses specific types of material for the printing process. In the following, a brief introduction to each AM group is provided: vat polymerization (VP) is the very first AM method that can solidify liquid resin or paste it into a 3D part (17). For that, different types of sources can be applied to the solidification process, classified into mainly laser and light-based sources. For example, stereolithography apparatus (SLA), micro-stereolithography (mSLA), and two-photon polymerization (2PP) are very common laser-based processes in vat polymerization. Digital light processing (DLP) is also a type of VP technique that uses a light-based source to cure the entire layer at once. As a result, DLP is able to quickly produce objects and parts with high accuracy (18). In the second AM group, the material jetting (MJ) process is defined as a multimaterial AM technique so that several materials can be deposited through individually controlled nozzles. The inkjet process is a recognized bioprinting method in this group which is specifically employed for tissue and organ bioprinting. Laser-induced forward transfer (LIFT) is another AM approach in the MJ group that uses a nozzle-free jetting technique with the ability to fabricate 2D/3D shapes (19). Material extrusion (ME) can typically print with one or multiple nozzles. Accordingly, similar to the MJ technique, it can be applied to the multimaterial 3D printing process (20). The feed material and extrusion mechanism can be categorized into filament-based, screw-assisted, and direct ink writing (DIW) techniques. Fused filament fabrication (FFF) is a filament-based AM process with a wide range of applications in various domains in particular for prototyping (21). One good example of a screwbased technique is pellet additive manufacturing (PAM) where polymer pellets can directly be melted through the printing nozzle and create a 3D object (22). DIW is one of the most versatile AM processes and is suitable for biopolymers. It simply squeezes material, mostly hydrogels, through the nozzle to make 3D shapes (23). Sometimes it needs a post-process, like UV light, to solidify the final printed parts. The other three AM processes, namely binder jetting (BJ), powder bed fusion (PBF), and sheet lamination (SL), are being used relatively less for natural biopolymers (collagen, gelatin, alginate). In BJ technique, adhesive inks are used to bond powder particles into a so-called “green part.” In this process, the printing inks are selectively deposited onto a layer of powders, then create final 3D shapes layer-by-layer (24). PBF also uses powder in the printing process. In particular, selective laser sintering (SLS) applies laser energy to fuse the polymer particles at the surface of a thin layer of powder, which is below the melting temperature. A roller spreads another thin layer of powder over the part bed. The process repeats until the 3D part is complete (25). The SL process is the last group of AM techniques for biopolymers that uses cutting and bonding processes. The feedstock is usually a layer of a material sheet bonded to the previous layer by glue or other adhesive material. After this, a laser or blade cuts it into a design shape. Chapter 2 overviews all these six AM techniques with more details as well as some practical examples of each technique.

Additive manufacturing of biopolymers

Currently, there is a wide range of applications for additively manufactured biopolymers products across different sectors (26). However, it significantly depends on the type of AM technique and the biopolymer. For example, alginate, gelatin, or collagen are typical hydrogels for bioprinting with many biomedical and pharmaceutical applications (27). This is because of their outstanding properties such as biocompatibility and biodegradability for wound healing, cartilage repair, and drug delivery (28). In another example, PLA and PCL are two common biopolymers with various biomedical applications. For instance, PLA can be used for pins and screws for fracture fixations (29), porous scaffolds (30), and automobile applications (31). Likewise, PCL can be used for the 3D printing of scaffolds for tissue engineering applications (32) or wound dressing (33) thanks to its biocompatibility properties. You will find several examples of each type of AM technique and biopolymers in the following chapters in more detail.

5. Challenges and future trends Although AM is a promising technology for the fabrication of biopolymers, there are still some challenges in the development of biopolymers related to their low inherent properties and their higher production cost, compared with conventional petroleum-based polymers. At the material innovation level, more research is needed into exploring the performance that can be achieved by adding structure via 3D printing to biopolymer building blocks. Research is advancing on mimicking new biopolymer-based materials for high-performance applications. Some biopolymers (such as gelatin and alginate) have been successfully used in 3D printing. However, biopolymer-based ink formulations are still rather finite and new inks need to be invented to address printability and material performance challenges and to meet diverse application needs. To develop real products, it is necessary to find out the actual thermomechanical and functional conditions for a particular application. As a result, materials specialists must adapt the properties of the biopolymers used in printing to ensure that these parameters are properly met. Regarding fabrication processes, almost all polymer-based AM techniques are, in principle, applicable to processing synthetic biopolymers but are not so easily adaptable for printing natural biopolymers. When considering cell incorporation during printing, generally only liquid-based polymer AM methods such as VP, MJ and DIW are currently considered suitable. There are still challenges to printing biopolymer hydrogels into complex structures (such as human organs or tissues) or constructs that can change with time or under specific stimuli (for 4D printing technology), which requires more research input. At the process level, more studies are needed on the life-cycle impact of a range of printable biopolymers, considering extraction and biopolymer processing, as well as printing and end-of-life disposal. To provide a more holistic picture of 3D printing

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biopolymers, LCA studies of biopolymer production need to be combined with 3D printing process LCAs, and end-of-life disposal options. Moreover, it is essential to have a comprehensive study on the recycling of 3D printed biopolymer-based parts, through developing processes like depolymerization and repolymerization. Overall, AM process innovation specifically for biopolymers should be a key focus area for development in the future so that the range of suitable biopolymers for each of the AM processes can be broadened and that more multi-material AM processes capable of printing dissimilar materials forms and types are enabled. Since AM is a highly multidisciplinary field, a broader collaboration between materials scientists, (polymer) chemists, mechanical engineers, computer scientists, and adjacent disciplines is required to provide sustainable, biobased, and fully circular alternatives. Biopolymers suitable for 3D printing are under continuous study to be used alone or in combination with other materials, drugs, and active compounds. The possibility to obtain them from renewable sources makes their use very promising in the future depicted by a reduced impact on the environment. This book explores various biopolymers that are currently used in additive manufacturing processes and highlights issues with/limitations in the materials and printing techniques. The purpose of this book is to consolidate the most recent knowledge on “materials” and “techniques” for the 3D printing of biopolymers, making it an excellent resource for everyone from students and academic researchers to business sectors. Furthermore, each chapter includes a special section devoted to future trends and prospects in the additive manufacturing of biopolymers from the perspectives of using biopolymers and new methodologies. The following paragraph introduces the chapters, presented in the book, which discusses basic elements and advancements in the additive manufacturing of biopolymers. In Chapter 2, additive manufacturing processes are described with a focus on their specification for the 3D printing of biopolymers. Also, useful information related to the printing of newly developed biopolymers is provided. Chapter 3 focuses on recent advances in both natural and synthesized biopolymers used in additive manufacturing by considering material design, printability, and their potential applications. In Chapter 4, 3D printing techniques for biobased hydrogels and the benefits and limitations of each 3D printing technique are detailed. Chapter 5 presents the different strategies to improve the fire retardancy of biobased 3D printed polymers, with a special focus on biobased PLA and PA11. Chapter 6 is dedicated to the additive manufacturing of biopolymer composites and nanocomposites. In Chapter 7, 3D printing of shape-switching biopolymers and their applications is described. In Chapter 8, the state-of-the-art of four-dimensional (4D) printing of biopolymers is given followed by some examples from recent research work in this area. Chapter 9 will present some post-processing techniques that can help to improve the mechanical and surface roughness after the 3D printing process. In Chapter 10, the 3D printing methods of biobased polymers and hydrogels used for tissue

Additive manufacturing of biopolymers

engineering are discussed. Chapter 11 is dedicated to the 3D printing of biopolymers for medical applications and devices and the associated moving regulatory issues in the US and the European Union (EU). In Chapter 12, the potential applications, and conditions of 3D and 4D printing of biopolymers with a special focus on biomedical applications are given. Finally, Chapter 13 discusses the different points regarding 3D printing, circular economy, and how AM techniques should be adapted to reach circular economy goals.

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20. Mehrpouya, M.; Vahabi, H.; Barletta, M.; Laheurte, P.; Langlois, V. Additive Manufacturing of Polyhydroxyalkanoates (PHAs) Biopolymers: Materials, Printing Techniques, and Applications. Mater. Sci. Eng. C 2021, 112216. 21. Barletta, M.; Gisario, A.; Mehrpouya, M. 4D Printing of Shape Memory Polylactic Acid (PLA) Components: Investigating the Role of the Operational Parameters in Fused Deposition Modelling (FDM). J. Manuf. Process. 2021, 61, 473e480. 22. Pricci, A.; de Tullio, M. D.; Percoco, G. Analytical and Numerical Models of Thermoplastics: A Review Aimed to Pellet Extrusion-Based Additive Manufacturing. Polymers 2021, 13 (18), 3160. 23. Li, L.; Lin, Q.; Tang, M.; Duncan, A. J.; Ke, C. Advanced Polymer Designs for Direct-Ink-Write 3D Printing. Chem.-Eur. J. 2019, 25 (46), 10768e10781. 24. Mostafaei, A.; Elliott, A. M.; Barnes, J. E.; Li, F.; Tan, W.; Cramer, C. L.; Nandwana, P.; Chmielus, M. Binder Jet 3D PrintingdProcess Parameters, Materials, Properties, Modeling, and Challenges. Prog. Mater. Sci. 2021, 119, 100707. 25. Shishkovsky, I.; Scherbakov, V. Selective Laser Sintering of Biopolymers with Micro and Nano Ceramic Additives for Medicine. Phys. Procedia 2012, 39, 491e499. 26. Li, N.; Qiao, D.; Zhao, S.; Lin, Q.; Zhang, B.; Xie, F. 3D Printing to Innovate Biopolymer Materials for Demanding Applications: A Review. Mater. Today Chem. 2021, 20, 100459. 27. Luo, Y.; Li, Y.; Qin, X.; Wa, Q. 3D Printing of Concentrated Alginate/Gelatin Scaffolds with Homogeneous Nano Apatite Coating for Bone Tissue Engineering. Mater. Des. 2018, 146, 12e19. 28. Pradhan, R.; Rahman, S.; Qureshi, A.; Ullah, A. Biopolymers: Opportunities and Challenges for 3D Printing. Biopolym. Ind. Appl. 2021, 281e303. 29. Middleton, J. C.; Tipton, A. J. Synthetic Biodegradable Polymers as Orthopedic Devices. Biomaterials 2000, 21 (23), 2335e2346. 30. Senatov, F. S.; Niaza, K. V.; Zadorozhnyy, M. Y.; Maksimkin, A.; Kaloshkin, S.; Estrin, Y. Mechanical Properties and Shape Memory Effect of 3D-Printed PLA-Based Porous Scaffolds. J. Mech. Behav. Biomed. Mater. 2016, 57, 139e148. 31. Tian, X.; Liu, T.; Yang, C.; Wang, Q.; Li, D. Interface and Performance of 3D Printed Continuous Carbon Fiber Reinforced PLA Composites. Compos. Appl. Sci. Manuf. 2016, 88, 198e205. 32. Wang, W.; Caetano, G.; Ambler, W. S.; Blaker, J. J.; Frade, M. A.; Mandal, P.; Diver, C.; B
artolo, P. Enhancing the Hydrophilicity and Cell Attachment of 3D Printed PCL/Graphene Scaffolds for Bone Tissue Engineering. Materials 2016, 9 (12), 992. 33. Muwaffak, Z.; Goyanes, A.; Clark, V.; Basit, A. W.; Hilton, S. T.; Gaisford, S. Patient-Specific 3D Scanned and 3D Printed Antimicrobial Polycaprolactone Wound Dressings. Int. J. Pharm. 2017, 527 (1e2), 161e170.

CHAPTER 2

Additive manufacturing and 3D printing techniques for biopolymers Jia An and Kah Fai Leong Singapore Centre for 3D Printing, School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore

1. Introduction There are three fundamental manufacturing processes: formative fabrication processes, subtractive fabrication processes, and additive fabrication processes (1). In the formative fabrication process, the desired shape is made by applying mechanical forces or restricting form to a body of raw materials to attain the desired shape. Examples are forging, bending, and injection moulding. In subtractive fabrication processes, the desired shape is made by selective removal of material until the final shape is achieved. Examples include milling, drilling, and electrical discharge machining. In the additive fabrication process, the desired shape is made by the selective addition of material, the opposite of the subtractive fabrication process. Examples are lamination and additive manufacturing (AM). Two or three of these fundamental processes can be combined to form hybrid manufacturing processes to enable a whole new level of design and manufacturing (1). Having many historical terms such as additive fabrication, layer manufacturing, solid freeform fabrication, and rapid prototyping, AM is formally defined jointly by the International Organization for Standardization (ISO) and ASTM International (formerly known as the American Society for Testing and Materials) as a process of joining materials to make parts from 3D model data, usually layer by layer (2). 3D printing is also a popular term due to the ease of understanding by the masses and it is often used interchangeably with AM (1,2). The basic process chain of AM comprises five basic steps (see Fig. 2.1): (1) creating a Computer-Aided Design (CAD) model; (2) converting the CAD model, usually, to a .stl file (a file format native to the stereolithography CAD software created by 3D Systems); (3) checking the model in the .stl file and preparing build parameters; (4) slicing and printing; and (5) postprocessing (1). The CAD model can be manually created or reconstructed from other data sources, e.g., dicom files of ultrasound, Computer Tomography (CT), or Magnetic Resonance Imaging (MRI). Although the .stl format is the de facto standard for almost all 3D printers, there are alternative file formats such as .obj, .step, .vrml, .3mf, .amf, available for designs that require multiple colours, materials, or objects. Slicing and printing are fully automated in all printers, but some printers do enable the

Additive Manufacturing of Biopolymers ISBN 978-0-323-95151-7, https://doi.org/10.1016/B978-0-323-95151-7.00009-0

© 2023 Elsevier Inc. All rights reserved.

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Fig. 2.1 The basic AM process chain.

printing to be manually paused and resumed so that changing or inserting other materials is possible. Postprocessing refers to the cleaning of supports used in the fabrication process followed by necessary treatments that give the final part its desired microstructures and mechanical properties. Depending on the AM material used in the process, a materialspecific postprocessing method may be applied, for instance, post-curing for photopolymer parts, vapor smoothing for thermoplastic parts, and heat treatment for electronic, ceramic, and metallic parts. AM process as a whole can also be viewed as single-step AM or multi-step AM (2). Single-step AM suggests minimal postprocessing such as cleaning, while multi-step AM prints a green part or a near-net-shape part that requires substantial postprocessing such as debinding and sintering. Compared to conventional manufacturing, the AM process has many advantages such as higher design freedom, greater flexibility, better customization, and lower wastage. Besides, the AM process is also considered as a better sustainable production method owing to its lower impacts on economical, environmental and societal concerns (3). These include achieving reduced cost for low volume high value parts, enabling decentralized manufacturing with reduced waste production, a safer work environment and with improved working conditions. Biopolymers can be broadly classified as natural biopolymers and synthetic biopolymers. Natural biopolymers can be further grouped into three categories: (1) polypeptide or protein-based such as collagen, fibrin, gelatine, silk and keratin; (2) polysaccharide or sugar-based, examples are chitin, chitosan, alginate, cellulose, hyaluronic acid; and (3) polynucleotide or DNA-based (4). Natural biopolymers possess excellent bioactivity, biocompatibility, and biodegradability, but they lack processibility and adequate mechanical strength. Synthetic biopolymers are not naturally present in the human body. Some examples are materials commonly used in FDA approved medical devices, e.g., polycaprolactone, poly(lactide-co-glycolide), polylactic acid (PLA), and polyhydroxyalkanoates (PHA) (5,6). These materials are not only easier to process and form into various geometries and shapes and produced at manufacturing scale, but also their composition and mechanical properties can be fine-tuned to address different application needs

Additive manufacturing and 3D printing techniques for biopolymers

such as biodegradable scaffolds and controlled release devices. However, they usually lack an adhesion mechanism to cell surface proteins and saccharides, presenting a combability issue at the cell-material interface. The topic of biopolymers will be discussed in detail in other chapters of the book. This chapter will focus on current AM techniques available for biopolymers. From the perspective of AM, there have been enormous research and application interests in AM of biopolymers as biopolymers are a new class of materials, and most are yet to become printable at the desired resolution, accuracy, quality, and consistency. How to allow biopolymers, especially natural biopolymers, be able to take full advantage of AM remains a significant challenge. Material modifications and process innovations are usually necessary to address the challenges of material-process limitations. In the case of material modification, the basic principle is that the improvement in biopolymer printability and characteristics should not compromise biological responses such as cell viability or cytotoxicity. For process innovation, the new or novel AM method should ideally be applicable to a wide range of materials rather than limited to a very specific biopolymer. Most of the current literature focuses on the material modification approach, while not much attention has been paid to the approach of process innovation. Therefore, it is necessary to analyze and discuss the key processes of current AM suitable for processing biopolymers. It is anticipated that some inspirations could be drawn to spark the development of great concepts for future AM of biopolymers.

2. Vat photopolymerization Photopolymerization is the very first commercial fabrication process employed in AM to solidify liquid or paste material into a 3D object. In most cases, the liquid or paste material is held in a container or vat for photocuring, hence it is termed vat photopolymerization. While in several other cases, liquid materials may be extruded as a filament or deposited in droplets from a nozzle (or multiple nozzles), and followed by ultra-violet (UV) or light curing. These so-called “vatless photopolymerization” techniques are part of other AM process categories of material extrusion and material jetting, which will be discussed in later sections. Vat photopolymerization can be further classified as laser beam-based or light pattern-based. 2.1 Laser beam-based Laser beam-based vat photopolymerization can be grouped into a single laser beam, multi-laser beams, and intersecting laser beams. Examples of single laser beam processes include stereolithography apparatus (SLA), micro-stereolithography (mSLA), and twophoton polymerization (2PP). SLA is invented in the late 1980s and is the first and classic commercial AM method. The process involves using a UV laser beam (e.g., 200 mm beam diameter and at 365 nm wavelength) to scan across the surface of a photopolymer

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Fig. 2.2 Laser beam-based vat photopolymerization (VP). (A) SLA; (B) inverted SLA; (C) 2PP; and (D) holography.

resin and selectively solidify a sliced contour of a CAD model (Fig. 2.2A). The photocured layer is lowered by one layer thickness, typically in the range of 20e100 mm, then a recoater sweeps to clean and smoothen the resin surface to be ready for the next scan. Layer by layer, a solid part is formed within the vat. A variation of the SLA method is by turning the classic method upside down (Fig. 2.2B). The UV laser scans the bottom of the vat through a clear transparent window and the cured layer moves up to allow liquid resin flowing back to refill the resulting gap. Then the cured layer moves down to one layer distance to the bottom, the UV laser repeats the scanning, and layer by layer, a solid part is formed, but with most part of it outside the vat. Two major advantages of inverted SLA are material savings and minimal support; however, maximum part size and part weight are compromised compared to the original SLA. When the laser beam diameter and layer thickness are significantly reduced (e.g., 7.5 and 10 mm, respectively), the system is called mSLA (7), which can print fine parts at a much higher resolution. Both SLA and mSLA rely on single photon absorption to initiate photopolymerization. To further improve printing resolution beyond mSLA, a slightly different principle is used. 2PP is a photopolymerization process in which the initiation of polymerization requires simultaneous absorption of two lower energy photons (e.g., energy of two infrared photons ¼ energy of one UV photon) (8). By focusing on an

Additive manufacturing and 3D printing techniques for biopolymers

ultrafast laser beam such as a femtosecond infrared laser, the photon flux reaches its maximum at the focal point, increasing the probability of simultaneous absorption (in contrast to sequential absorption) of two infrared photons (Fig. 2.2C). Subsequently, this triggers an immediate photopolymerization and solidification at the focal point, but not anywhere else. In 2PP, support is not required due to the small scale and ultrafine structures and features as small as 100 nm are achievable. These features can be arbitrarily printed rather than printing layer by layer. However, the major sacrifices of gaining ultrahigh resolution are part size and print speed. This problem is yet to be overcome by combing 2PP with SLA in a single setup. This could be one of the research directions in process innovation. The motivation for using multiple laser beams in SLA is to increase print speed. In one approach, two wavelengths, 405 nm (blue light) and 532 nm (green light) were combined in SLA in 2010 (9,10). The blue light laser, owning to its curing resolution, was used to scan contours or outlines. The green light, for its high power and wide cured width, was used for internal crosshatch curing. The combination of two lasers significantly speeds up part fabrication, especially when there are volumetric solid features. In a more straightforward approach, 3D Systems launched SLA 750 Dual - an SLA system installed with two laser beams in 2022 (11). Obviously, the print speed can be easily doubled by properly coordinating synchronously the two curing spots and 3D Systems claims that their system is able to triple the throughput of other available stereolithography solutions. Nevertheless, there is no intersection of these laser beams in this system. The development of systems with intersecting laser beams is driven by the concept of “volumetric AM,” a term frequently used to describe a group of recent AM processes that attempts to print a whole part directly without using support and not necessarily in a layer-by-layer manner. One such example is a holography-based 3D printing reported in 2017 (12). In this method, a low concentration of photoinitiator was used and three laser beams of the same wavelength from x, y, and z directions were directed to precisely intersect at an exact desired location, producing constructive interference such that the resin at the superposition point can absorb sufficient energy to reach curing thresholds of the resin (Fig. 2.2D). Like 2PP, there is no curing elsewhere during the process and the curing voxel can be moved in any direction within the resin volume. 2.2 Light pattern-based Laser beam projects a tiny spot and it always takes time for the spot to complete scanning an area. Therefore, projecting a light pattern that cures an area instantly without scanning time is the main principle behind all light pattern-based vat photopolymerization (VP) techniques. Digital light processing (DLP) is one of the early methods that use projected light patterns to cure photopolymer. To project a light pattern, there are two basic requirements. First, the sliced layer of an STL model must be converted to a grayscale image consisting of pixels. Secondly, the pixel pattern must be converted to the light

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pattern. Thanks to the invention of the digital micromirror device (DMD), a chip with micromirror arrays on its surface and each micromirror measured 13.7 mm  13.7 mm in size corresponding to a pixel, the pixel information of a contour image can be converted to a light pattern by controlling individual micromirrors to either reflect light toward a screen or tilt it away from a screen. With the DMD chip, a light pattern of a crosssectional contour can be projected onto photopolymer resin for immediate curing. As the light projector is positioned right below the vat, it is always the bottom layer being cured according to the light pattern (Fig. 2.3A). After one light exposure, the cured layer moves up to allow the liquid resin to refill and be readied for curing for the next layer. Like inverted SLA, the final part is formed outside the vat, and only a small amount of photopolymer and minimal support are required. Besides DMD, a comparatively less expensive liquid crystal diode (LCD) panel is another alternative device that can convert

Fig. 2.3 Light pattern-based VP. (A) DLP; (B) MSLA; (C) CLIP; (D) HARP.

Additive manufacturing and 3D printing techniques for biopolymers

the pixel information of an image into a light pattern. However, they may also require more maintenance and upkeep. Masked stereolithography (MSLA) or LCD SLA is the method of using an LCD panel (connected to a computer) to generate a pixel-based mask, which filters the incoming light source into a light pattern (Fig. 2.3B) (13). One advantage of using LCD is the control of grayscale pixels (in contrast to binary black and white) to modulate the degree of photopolymerization and has the capability to cure functionally graded structures. However, most of the photopolymers are cured in the UV range, and using UV as a light source can shorten the lifespan of LCD. This is the mask module becomes a consumable item. Although DLP and MSLA can do away with scanning time, there is still a significant amount of time needed to move the cured layer up and down during the printing process. Continuous liquid interface production (CLIP) is therefore proposed and patented in 2014 to eliminate the waiting time for movements between layers (14). To enable continuous upward movement of a cured layer, the cured layer must be completely free from attaching to the base. Oxygen is known to have an inhibition effect on free radical polymerization and therefore creating an oxygen-rich layer separating the cured layer and the base is the key underwriting principle of CLIP (14). When oxygen diffuses through the transparent and gas-permeable base, a “dead layer” (oxygen-rich layer) with controlled thickness can gradually build up at the base (Fig. 2.3C). Projected light pattern passes through the “dead layer” but without curing it due to oxygen inhibition. Instead, the light pattern cures a layer above the “dead layer,” thus creating a cured layer above a liquid layer. Since the solidified layer is free from the base, it can then be continuously pulled up by synchronizing the printing speed to the curing speed. Inserting a middle layer is an interesting concept and high area rapid printing (HARP) is another example of this approach introduced in 2019 (15). In this method, a flowing layer of immiscible oil is used to separate the cured layer from the base (Fig. 2.3D). The oil is fluorinated to reduce the adhesive forces at the interface during circulation and at the same time, it can dissipate the heat generated from photopolymerization, allowing for a larger print area. Light pattern-based VP has also been explored for volumetric AM. One example is computed axial lithography (CAL) reported in 2019 (16). This method is inspired by the image reconstruction procedures of CT, in which a 3D virtual model can be created from the superposition of images taken from multiple different angles. Likewise, when light patterns of these images from multiple angles are projected into the photopolymer resin, a superposed 3D physical model can be formed (Fig. 2.4A). Although each image projection propagates through the resin, it is not sufficient to solidify the material. Only the superposed volume receives sufficient energy for solidification. The superposition of the images is realized by projecting a video synchronizing to the rotating resin. Besides being support-free, one major advantage of CAL is to fabricate microdevices using extremely viscous materials such as silica glass nanocomposites (17). Another example

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Fig. 2.4 Light pattern-based volumetric AM. (A) CAL and (B) xolography.

of light pattern-based volumetric AM is xylography reported in 2020 (18). In this method, an important concept is a “light sheet,” which is “a thin layer of light” diverged from a single laser beam through a cylindrical lens (Fig. 2.4B). The light sheet first excites a thin layer of photoinitiators into a latent state (not yet to trigger polymerization), then an orthogonally arranged projector generates a light pattern onto the light sheet to solidify one layer. “Photoswitchable photoinitiator” is another important concept in this method, which refers to the fact that only photoinitiators in the latent state absorb the second light and trigger polymerization. As the light sheet plane is moving linearly relative to the vat, the projector can play a video synchronized to the moving speed so that a 3D object is printed continuously without any waiting time between layers. In general, biopolymers are mostly not compatible with the above VP processes. Researchers have been focusing on their derivatives for photocuring and printability. Some examples of the derivatives are gelatin methacrylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, and PEGylated fibrinogen (19). Table 2.1 shows more examples of biopolymer derivatives that have been developed for the VP processes. These photocurable biopolymers may be blended for multi-functional applications or used as a matrix for nanocomposites. Aside from material modifications, the number of materials printable is also of great interest. For example, there are already several attempts on developing multi-material vat photopolymerization (40e42). Nevertheless, multi-material fabrication is still a challenge in VP because multi-material fabrication requires precise allocation of different components or compositions within a single layer, rather than distributing materials in alternate layers.

Additive manufacturing and 3D printing techniques for biopolymers

Table 2.1 A summary of examples of photocurable biopolymers suitable for VP. Photocurable biopolymer

References

Gelatin methacrylate (GelMA) Chondroitin sulfate methacrylate (CSMA) Fibrinogen, PEGylated Hyaluronic acid methacrylate (HAMA) Silk fibroin methacrylate (SF-MA or SiMA) Chitosan methacrylate Fetal bovine albumin methacryloyl (BSA-MA) Alginate methacrylate Pectin methacrylate (PECMA) Oxidized hydroxypropyl cellulose methacrylate (Ox-HPC-MA) Poly(ethylene glycol) diacrylate (PEGDA) poly(ethylene glycol) methacrylate (PEGMA) Methacrylate cardan phenolic polycondensate (MCPP) Poly(methyl methacrylate) (PMMA) Glycidyl methacrylate (GMA) 4-arm polycaprolactone methacrylate (4PCLMA) Polylactic acid methacrylate Polyurethane methacrylate Poly(dimethylsiloxane) methacrylate

(20) (21) (22) (23) (24,25) (26,27) (28) (29) (30) (31) (25) (32) (33) (34) (35) (36) (37) (38) (39)

3. Material jetting Unlike VP, material jetting is a multi-material AM process and different materials can be deposited through individually controlled nozzles. Depending on the material, it can be grouped into liquid drop jetting, paste drop jetting, melt drop jetting, and metal drop jetting. 3.1 Liquid drop jetting Liquid drop jetting is the most common technique in material jetting. AM methods based on liquid drop jetting can be further subdivided into conventional inkjets, aerosol jets, and electrostatic jets. Examples of conventional inkjet include thermal inkjet and piezoelectric inkjet (Fig. 2.5A). In a thermal inkjet, the liquid inside the nozzle is compressed by a heat-induced gas bubble to eject one droplet (typically 20 mm in size), while in a piezoelectric inkjet, the deformation of piezoelectric material compresses the liquid to eject one droplet. It should be noted that generating a droplet alone is not yet AM, there is another critical requirement - solidification of the droplet. Photopolymers are liquid and solidify upon UV exposure, therefore they are usually selected as the material of choice for material jetting AM. PolyJet is a representative photopolymer-based inkjet technology. The printhead consists of arrays of nozzles, depositing both model materials and support materials during printing, which are immediately photocured by the UV

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Fig. 2.5 Material jetting. (A) Conventional inkjet; (B) aerosol jet; (C) electrostatic jet; (D) LIFT; (E) melt jet; and (F) metal jet.

lamps installed on both sides of the printheads. The recent model of PolyJet (Stratasys J850), launched in 2021, can print multiple materials and multiple colours in a single build, including transparent material. Another interesting inkjet-based AM technology is nanoparticle jetting (NPJ) by XJet released in 2019. It is designed to print both ceramic and metallic suspensions to complement Polyjet materials. Besides photopolymer, ceramic and metallic nanoparticles, inkjet AM can also co-print reactive materials (i.e., reactive jetting) for high viscosity and high-performance applications (43,44). Compared to inkjet, aerosol jet, first patented and commercialized by Optomec Inc. in 2004, results in smaller droplet size and hence able to attain finer resolution (45). The raw liquid is first atomized into a mist of aerosol by ultrasound or high pressure, then the aerosol is condensed and directed toward a nozzle where the inner aerosol is accelerated by the outer sheath gas to generate a stream of collimated tiny aerosol droplets (1e5 mm)

Additive manufacturing and 3D printing techniques for biopolymers

(Fig. 2.5B). Depending on the materials used, the mechanism of solidification could be photocuring, however, the primary use of aerosol jet is to print meso-scale electronic devices such as sensors and connectors, in which metallic nanoparticles are deposited in loose adherence first and then sintered into a solid pattern. Owing to a unique design, the aerosol jet nozzle is free from clogging compared to the inkjet nozzle and it can process liquid at a wider range of viscosity. Most of the electrostatic jetting AM are modified from conventional electrospinning by implementing an x-y-z framework to the nozzle, and a few new terms have been derived such as near-field electrospinning, melt electro-writing and electrohydrodynamic jet printing (E-jet) (46e48). In these methods, the electrostatic charges build up at the liquid drop surface, generating repelling force to counter surface tension and eventually break it, resulting in a jet of tiny droplets (Fig. 2.5C). Special electrostatic jet technology is a superfine inkjet printer developed by SIJ Technology (Japan) in 2018 (49). Although called inkjet, the ejection is based on electrostatic force. This technology can deposit a single droplet less than 1 mm in diameter. When used for 3D printing applications, microstructures at a scale close to 2PP can be fabricated. However, compared to its excellent capability of 2D patterning, the potential of microscale multi-material 3D printing still needs to be further explored. 3.2 Paste drop jetting Many natural biopolymers are in the form of paste or hydrogel and are generally not ideal for nozzle-based jetting. Nozzle-free jetting is therefore of considerable interest to processing natural biopolymers. Laser-induced forward transfer (LIFT) is one such nozzlefree jetting technique capable of 2D patterning and making some simple 3D shapes (50). First introduced in 1986 it found a new impetus for development in 2016. The setup of LIFT requires a transparent donor substrate, having a very thin layer of material (a few micrometers) beneath it, and a receiver substrate up to one or 2 mm away from the donor (Fig. 2.5D). A pulsed laser rapidly heats the interface between the donor and thin film, generating pressure within the laser-irradiated area, and the pressurized bubble mechanically breaks the thin film, releasing the material to the receiver substrate. Paste and hydrogel materials in thin layers can be ejected by this method. One major advantage of LIFT is the high printing speed and fine resolution. However, the gap between donor and receiver is small, and its potential in creating complex 3D structures is yet to be fully realized. Furthermore, the use of laser makes such printers comparatively more expensive than most nozzle-based printers. 3.3 Melt drop jetting There are few AM processes that use polymer melt drop jetting. Melt electro-writing is one example as the aforementioned in electrostatic jetting. It uses high voltage and electrostatic force to deposit melt drops but eventually, the melt drops are stretched into

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microfibers rather than landing at a substrate in droplet form. The other example is Arburg’s Free Former process (51). In this method, polymer pellets are first fed into the screw extruder for heating and mixing, and then the melt is injected into a nozzle where the nozzle closure punches out one melt drop (Fig. 2.5E). The melt drop solidifies upon natural cooling, and a part can be printed drop-by-drop. Obviously, melt dropbased printing is a slow process compared to filament-based extrusion. However, Free Former can print a filament drop-by-drop at a controlled deposition rate, which means that this method is able to control the microstructure of a single filament - a unique feature of droplet jetting. This can result in many part properties (e.g., isotropy, porous filament, droplet density-based porosity) that cannot be possibly achieved by filamentbased extrusion. 3.4 Metal drop jetting Compared to other material jetting processes, a typical metal drop jetting printer also requires a pulsing plunger to punch out molten metal droplets (52). However, the droplet is charged upon exiting so that the direction of the droplet can be controlled via an electric field, usually at a small deflection angle during printing (Fig. 2.5F). Although the interdroplet interface may lack sufficient metallic bonding due to the stacking of droplets, the final part strength can be improved by further postprocessing such as sintering. Alternatively, the metal droplet may also be generated by a new method called magnetohydrodynamic jetting (53). In this method, the Lorentz force induced by the current and magnetic field is used to eject a metal drop. While metal drop jetting may not be highly relevant to biopolymer AM, its similarity with melt jet can inspire new developments in melt drop jetting in the future. Table 2.2 shows several examples of MJ-based AM of biopolymers. There have been a growing number of researches using the MJ-based AM process, mainly in the areas of Table 2.2 Some examples of MJ-based AM of biopolymers. Process

Material

Application

References

Inkjet Inkjet Inkjet Inkjet Aerosol jet Aerosol jet Electrohydrodynamic Electrohydrodynamic LIFT LIFT

Alginate Polyethylene glycol Silk fibroin, alginate Collagen, fibrinogen, thrombin Collagen Gelatine Polycaprolactone Polycaprolactone Alginate Hyaluronic acid, methylcellulose, alginate Polylactic acid

Drug delivery Microfluidics Bioprinting Bioprinting Bioprinting Bioprinting Drug delivery Retinal scaffold Bioprinting Bioprinting

(54) (55) (56) (57) (58) (59) (60) (61) (62) (63)

3D printing

(64)

Melt drop

Additive manufacturing and 3D printing techniques for biopolymers

tissue engineering, drug delivery and bioprinting. Inkjet has the key advantages of multimaterial printing and precise patterning. Many photocurable biopolymers developed for VP, in principle, could be explored in photocuring-based inkjet process for multibiomaterial jetting. However, material jettability, stackability in 3D, and nozzle clogging (typical for inkjet) are common issues and challenges in MJ-based AM. Solving these problems concurrently for MJ can be more difficult than simply just adding a methacrylatetion function to a polymer for VP. Besides inkjet, other MJ process are also suitable for printing biopolymers, but some are less popular for the reason of the high costs involved (e.g., LIFT). Melt drop printing is new even in the general field of AM, therefore many biopolymer pellets have yet to be trialed for this printing process.

4. Material extrusion Like material jetting, material extrusion can also be a multi-material AM process, and the number of materials on a typical printer depends on the number of nozzles available on the printer. Based on the feed materials and extrusion mechanisms, material extrusion can be grouped into filament-based, screw-assisted, and direct ink writing (DIW) material extrusion. 4.1 Filament-based A process that was first patented in 1989, filament-based extrusion process requires a spool of the filament of constant diameter and composition. There are several such systems and examples include fused deposition modeling (FDM), fused filament fabrication (FFF), and continuous fiber fabrication (CFF). In this technique, the filament is forwarded by two driving wheels into a liquefier, pushing the melt inside out of the orifice, and the moving nozzle stretches the melt into a thin filament (Fig. 2.6A) (65,66). The temperatures of nozzle, printing bed, and chamber ambiance influence the range of printable materials possible and part quality achievable. The printability of the filament can be determined by whether it buckles under the pushing forces by the driving wheels before entering the liquefier. In FDM and FFF, the nozzle stops extrusion at the end of each pass, while in CFF, due to the continuous fiber, a cutter is used to end each pass. Common synthetic biopolymers are well suited for this technique and recent developments have been paying attention to biopolymer composite filaments. 4.2 Pellet-based Pellet-based extrusion, also known as screw-assisted extrusion, works in an analogous way to filament-based extrusion except that the feedstock is in the form of polymer pellets without the need to convert to filament (Fig. 2.6B). There are large-format pellet printers as well as desktop pellet printers, for example, big area additive manufacturing (BAAM) and Pollen’s pellet additive manufacturing (PAM), respectively (67e69).

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Fig. 2.6 Material extrusion. (A) Filament-based; (B) screw-assisted; and (C) DIW.

BAAM can deposit materials at 50 kg per hour in a volume of 25 m3, while PAM can blend and print a wide range of polymers including high-performance polymers. Interestingly, PAM is also capable of printing ceramic parts using injection moulding ceramic pellets (70). 4.3 Direct ink writing First developed in 1996, DIW is one of the most versatile and the least expensive extrusion-based AM processes suitable for AM biopolymers; it is akin to and as simple as squeezing a tube of toothpaste. However, extrudability alone may not be considered AM, the extruded paste or hydrogel filament must stack in height with minimal deformation, that is, the structural integrity and shape fidelity are important in the context of 3D printing. This requirement for 3D shapes highlights two important properties of DIW inkdviscosity and thixotropy, provided there is no other physical or chemical crosslinking involved except that the ink supports its own weight. If alternative solidification is necessary, such as sol-gel transition, UV or ion crosslinking, the ink properties related to these mechanisms become more important, e.g., the degree of methylation will determine the tunable properties in some photocurable inks. The DIW process can be pneumatically or mechanically driven, and the extrusion of the filament can be dry or wet (Fig. 2.6C). Pluronic hydrogel has been used as a support to construct overhanging and hollow features on a dry platform (71), while in the case of FRESH - freeform reversible embedding of suspended hydrogels (72), the filament is extruded into a supporting bath to counter the weight so that overhanging and hollow features can be printed without collapse. The nozzle of DIW can be multiplied side by side for multimaterial printing or multi-phase printing (73). Interestingly, multiple nozzles can also

Additive manufacturing and 3D printing techniques for biopolymers

be arranged concentrically or co-axially such that dissimilar materials are co-extruded at a different rate for a variety of applications such as encapsulation and generating hollow tubes (74). The number of publications on co-axial printing of biopolymers and cells has been steadily growing in recent years as reviewed in (75,76), with a focus on encapsulation of cells and drugs for tissue engineering, drug delivery and disease modeling taking advantage of the core-shell structure provided by the co-axial nozzle. Besides the above-mentioned examples, there are several works using extrusionbased AM to print biopolymers for various applications. Table 2.3 shows a selection of these examples. The emerging applications in pharmaceutical printing and food printing are new growing areas in the field of AM, they can be of particular interest in AM of biopolymers.

5. Other solid-based AM processes Natural biopolymers are mostly made of polypeptides, polysaccharides, and polynucleotides. These materials are more suitable for liquid-based AM processes, hence VP, MJ, and ME are discussed more in detail as they are either liquid-based or applicable to liquid. The remaining four categories of AM, namely powder bed fusion (PBF), binder jetting (BJ), sheet lamination (SL), and directed energy deposition (DED), are primarily solidbased AM, they are less relevant to liquid forms of natural biopolymers in their current process and material handling capabilities, although some of these processes may be used for processing synthetic biopolymers and solid forms of natural biopolymers.

Table 2.3 A selection of examples of extrusion-based AM of biopolymers. Process

Material

Application

References

Filament Filament

Polycaprolactone Cellulose, poly(ethylene glycol), poly(vinyl alcohol) Polylactic acid and starch Polyether ether ketone Polycaprolactone Polyether ether ketone Biopolyesters Alginate Cellulose Gelatine Silk fibroin Collagen Starch

Tissue engineering scaffolds Pharmaceuticals

(77) (78)

3D printing Medical implants Tissue engineering scaffolds Medical implants 3D printing Drug delivery Antibacterial objects Bioprinting, food printing Tissue engineering Bioprinting Food printing

(79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89)

Filament Filament Pellet Pellet Pellet DIW DIW DIW DIW DIW DIW

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5.1 Powder bed fusion Powder bed fusion (PBF) is a process category that involves the use of a heat source to selectively fuse a part on a powder bed (Fig. 2.7A). There are generally two categories of powder bed fusion: polymer PBF and metal PBF (90). Examples of polymer PBF include selective laser sintering (SLS) and multi-jet fusion (MJF). In the SLS process, a roller spreads polymer powder into a thin layer, then a carbon dioxide laser scans the powder layer to draw a contour. Afterward, the solidified layer is lowered by one layer thickness and the roller spreads another thin layer of powder over the part bed. The process repeats until the part is complete. An inert gas may be needed in the chamber to prevent oxidation or explosion. SLS is based on the principle of solid-state sintering in which powder particles fuse together at the surface without the bulk undergoing melting. Therefore, the processing window for a semicrystalline polymer is the temperature range above the crystallization point but below the melting point. One example biopolymer suitable for SLS is polycaprolactone, which has been shown to be printable for tissue engineering scaffolds and drug delivery device applications (91e96). MJF is slightly different from SLS in that the solidified contour is not drawn by its heat source (i.e., infrared lamp), instead, inkjet nozzles deposit fusing agents and detailing agents to define where to fuse the powder and where to remain loose upon exposure to heat. The fusing agents promote the absorption of more infrared energy while the detailing agents inhibit it. As

Fig. 2.7 Other solid-based AM processes. (A) PBF; (B) BJ; (C) SL and (D) DED.

Additive manufacturing and 3D printing techniques for biopolymers

MJF requires inkjet agents and is not a fully open system, there is limited work on printing synthetic biopolymers. Selective laser melting (SLM) and electron beam melting (EBM) are typical examples of metal PBF. SLM uses a high-power fiber laser and an inert chamber to melt and print non-reflective metals. EBM requires a vacuum chamber, and its defocused electron beam is capable of postprint heat treatment compared to SLM. While they are generally not applicable to polymer powder due to high processing temperatures, there has been working on printing some metal-polymer hybrid structures that may have future applications for hybrid products with biopolymers (97,98). 5.2 Binder jetting (BJ) Binder jetting is a process category that uses adhesive inks to bond powder particles into a preliminary solid or green part. The adhesive inks are selectively deposited onto a thin layer of powder via inkjet printheads (Fig. 2.7B). Once in contact with a powder bed surface, the liquid inks quickly spread and infiltrate the powder layer driven by capillary forces. Although a higher powder packing density or binder saturation may help reduce porosity and increase part strength, it usually requires a secondary process such as infiltration or heating to reinforce the green part (99,100). One main advantage of BJ over PBF is low-temperature printing, which means a wider range of powder materials and there is little thermal gradient-induced deformation. As long as the powder is spreadable and there is a suitable binder (e.g., a solvent of the powder material), it could be printable by BJ. However, the majority interest of BJ is currently in metal printing, the potential of binder jetting for printing biopolymers has not yet been fully explored because unlike metals, there are many alternative low temperature and more convenient processing methods for biopolymers such as VP and DIW. In addition, BJ requires a large number of materials for printing due to the need for a large powder bed, which can be challenging for expensive biopolymers. Nevertheless, there is still many possibilities in the underexplored room for low-cost solid biopolymers. 5.3 Sheet lamination Sheet lamination (SL) is a process category that is based on the art of bonding and cutting. The feedstock is either a stack of standard sheets or a roll of sheet material. It works by bonding a layer of a sheet to the previous layer, usually by glue or other adhesives, and then cutting out a contour using a carbon dioxide laser or a tungsten blade (Fig. 2.7C). Outside each contour is a crosshatch cut, which layer-by-layer becomes cubes and blocks surrounding the final part and supporting it. After printing, the supports are removed to reveal the part inside. It is possible to print a colored part inside a white paper block as shown in selective deposition lamination (SDL) (101). SL is particularly interesting in AM biopolymers because the standard SL material is paper, which is mainly celluloseda natural biopolymer. For example, standard A4 paper has been used as a feedstock in the SDL process, and the final product is a solid part made from paper, mainly used for

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conceptual prototyping. However, it would be more relevant to biomedical applications if bacterial cellulose is densely packed and is made into a lattice structure for tissue engineering applications. The main challenge is to achieve a highly porous architecture and yet ensure the cellulose structs to be fully dense. 5.4 Directed energy deposition Directed energy deposition is a process category that uses an energy source of high heat input such as a laser, electron beam, and arc to melt either powder particles or a metal wire for printing (Fig. 2.7D). The molten puddle solidifies as the heat source is moving away. It is mainly used to build a near-net shape for large but moderately complex metallic parts. Materials can also be added to the existing surface for repair or adding new features. However, DED is the least relevant to biopolymers among all the AM processing categories. Given the requirement of a solid feedstock, not many biopolymers are suitable for PBF, BJ and SL. In addition, both powder bed and sheet laminate require a large number of materials for printing, which is not favorable for biopolymers that are generally more expensive in cost or limited in yield. Additional examples of solid biopolymer printing are shown in Table 2.4.

6. Bioprinting and hybrid biomanufacturing Bioprinting can be narrowly defined as the 3D printing of cell-laden materials and cell spheroids. The major bioprinting techniques are indeed those liquid-based AM methods that are compatible with mixing with cells, such as DIW, inkjet, LIFT, electrostatic jet, and VP. However, with over 2 decades of development, bioprinting has already demonstrated some interesting applications beyond its initial intended use in pharmacy and medicine, for example, cultured meat (110), plant science (111), bioprocessing (112), and space missions (113) (See Fig. 2.8). Therefore, bioprinting should be viewed Table 2.4 Some examples of biopolymers. Process

Material

Application

References

Selective laser sintering Selective laser sintering Selective laser sintering Selective laser sintering Selective laser sintering Binder jetting Binder jetting Binder jetting Sheet lamination

Polycaprolactone Poly(L-lactide) Poly (vinyl alcohol) Polyether ether ketone Starch Polycaprolactone Starch Cellulose Cellulose

Tissue engineering scaffolds Tissue engineering scaffolds Tissue engineering scaffolds Medical implants Food printing Drug delivery 3D printing Food printing Packaging

(95) (102) (103) (104) (105) (106) (107) (108) (109)

Additive manufacturing and 3D printing techniques for biopolymers

Fig. 2.8 Applications of bioprinting.

separately as a broad category in order to be studied more in-depth and up to date with its development. It is our opinion that bioprinting is an important emerging research area that will lead to the discovery and standardization of new and novel biomaterials and biological materials for all kinds of 3D printing processes and applications. Thus they may incorporate cells or living tissues or not and can be directed toward pharmaceutical, medical or a whole host of other applications. AM of biopolymers can therefore be considered as a form of bioprinting. Nevertheless, the necessities of incorporating cells in AM of natural biopolymers usually dominate the research as compared to those in AM of synthetic biopolymers that this, perhaps, contributes to the ambiguity of the differences between AM of biopolymers and bioprinting. Table 2.5 shows the feasibility-in-principle and perceived relevance when mapping the processing of natural and synthetic biopolymers to the available AM methods. The number of ticks represent perceived relevance. It is apparent that synthetic biopolymers are more processable compared to natural biopolymers. In addition, material modifications are necessary to make natural biopolymers to be

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Table 2.5 Overview of AM and 3D printing techniques for biopolymer. AM technique

VP MJ

ME

PBF BJ SL DED

Laser beam Light pattern Liquid drop Paste drop Melt drop Metal drop Filament Pellet Direct ink writing Polymer Metal Polymer Metal Polymer Metal Metal

Natural biopolymer

Synthetic biopolymer

OOO OOO OO OO e e O O OOO e e O e O e e

OOO OOO OO OO OO e OOO OOO OOO OOO e OO e OO e e

Legend: OOO, very relevant; OO, relevant; O, somewhat relevant, not relevant.

fully compatible with an AM process, while most synthetic biopolymers can be used easily and directly on the AM machines. Hybrid biomanufacturing refers to the combination of different 3D printing methods and/or non-3D printing methods into one printing system so that new properties of the printed part can be realized by the complementary processes. A typical example is a multi-head bioprinter consisting of an inkjet printhead, extrusion printhead, electrospinning tool head, and post-curing tool head (114). Since a few biopolymers and AM techniques have been technically matched to work, for example, the aforementioned photocurable biopolymers and biopolymers printable by various other AM processes, it is interesting to explore in the future what types of combinations will result in the most advanced functionalities beyond just on shape and geometry.

7. Conclusions and future perspectives In this chapter, AM and 3D printing techniques for biopolymers are discussed from the perspective of the AM process categories. In VP, the current focus is on developing photocurable biopolymers and methods for printing multi-biopolymers. In MJ and ME, which are well capable of multi-material printing, combining both printing modes in a hybrid bioprinter is of current interest. In PBF, the range of biopolymers is currently limited to solid synthetic biopolymers due to laser processing, therefore developing biopolymer nanocomposites is one good way forward in its development. In BJ, which

Additive manufacturing and 3D printing techniques for biopolymers

is a low-temperature process, is well suited for printing pharmaceuticals and food materials. In SL, the current relevant biopolymer is cellulose. Overall, almost all polymer AM methods are, in principle, applicable for processing synthetic biopolymers but are not so easily adaptable for printing natural biopolymers. When considering cell incorporation during printing, generally only liquid-based polymer AM methods such as VP, MJ and DIW are currently considered suitable. Regarding the applications of AM of liquid-based biopolymers, the majority of the applications are concentrated on tissue engineering, bioprinting and drug delivery. In solid-based biopolymers, exploring the effects of diverse nanomaterials and extending biomedical applications to new areas such as pharmacy and food are fast growing. Moreover, the fundamental issue in AM of biopolymers is material-process combability. It is suggested that it may not necessarily always rely on the improvement of material printability to solve the challenge. AM process innovation specifically for biopolymers should be a key focus area for development in the future so that the range of suitable biopolymers for each of the AM processes can be broadened and that more multi-material AM processes capable of printing dissimilar materials forms and types are enabled. Furthermore, our understanding of bioprinting should be more inclusive to keep up with rapid changes and new developments in the area. Hybrid biomanufacturing is an interesting trend worth watching in the future because it involves the integration of state-of-theart 3D printing and associated technologies toward the automated, single-step fabrication of multi-functional bio-constructs.

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71. Suntornnond, R.; Tan, E. Y. S.; An, J.; Chua, C. K. A Highly Printable and Biocompatible Hydrogel Composite for Direct Printing of Soft and Perfusable Vasculature-Like Structures. Sci. Rep. 2017, 7 (1), 1e11. 72. Mirdamadi, E.; Tashman, J. W.; Shiwarski, D. J.; Palchesko, R. N.; Feinberg, A. W. FRESH 3D Bioprinting a Full-Size Model of the Human Heart. ACS Biomater. Sci. Eng. 2020, 6 (11), 6453e6459. 73. Saadi, M.; Maguire, A.; Pottackal, N.; et al. Direct Ink Writing: A 3D Printing Technology for Diverse Materials. Adv. Mater. 2022, 34 (28), 2108855. 74. Liu, W.; Zhong, Z.; Hu, N.; Zhou, Y.; Maggio, L.; Miri, A. K.; Fragasso, A.; Jin, X.; Khademhosseini, A.; Zhang, Y. S. Coaxial Extrusion Bioprinting of 3D Microfibrous Constructs with Cell-Favorable Gelatin Methacryloyl Microenvironments. Biofabrication 2018, 10 (2), 024102. 75. Mohan, T. S.; Datta, P.; Nesaei, S.; et al. 3D Coaxial Bioprinting: Process Mechanisms, Bioinks and Applications. Prog. Biomed. Eng. 2022, 4 (2), 022003. 76. Rafiee, M.; Granier, F.; Therriault, D. Advances in Coaxial Additive Manufacturing and Applications. Adv. Mater. Technol. 2021, 6 (11), 2100356. 77. Meng, Z.; He, J.; Cai, Z.; Wang, F.; Zhang, J.; Wang, L.; Ling, R.; Li, D. Design and Additive Manufacturing of Flexible Polycaprolactone Scaffolds with Highly-Tunable Mechanical Properties for Soft Tissue Engineering. Mater. Des. 2020, 189, 108508. 78. Melocchi, A.; Parietti, F.; Maroni, A.; Foppoli, A.; Gazzaniga, A.; Zema, L. Hot-Melt Extruded Filaments Based on Pharmaceutical Grade Polymers for 3D Printing by Fused Deposition Modeling. Int. J. Pharm. 2016, 509 (1e2), 255e263. 79. Hary
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92. Sudarmadji, N.; Tan, J. Y.; Leong, K. F.; Chua, C. K.; Loh, Y. T. Investigation of the Mechanical Properties and Porosity Relationships in Selective Laser-Sintered Polyhedral for Functionally Graded Scaffolds. Acta Biomater. 2011, 7 (2), 530e537. 93. Leong, K. F.; Wiria, F. E.; Chua, C. K.; Li, S. H. Characterization of a Poly-ε-Caprolactone Polymeric Drug Delivery Device Built by Selective Laser Sintering. Bio Med. Mater. Eng. 2007, 17 (3), 147e157. 94. Leong, K. F.; Chua, C. K.; Gui, W. S. Building Porous Biopolymeric Microstructures for Controlled Drug Delivery Devices Using Selective Laser Sintering. Int. J. Adv. Manuf. Technol. 2006, 31 (5), 483e489. 95. Yeong, W. Y.; Sudarmadji, N.; Yu, H. Y.; Chua, C. K.; Leong, K. F.; Venkatraman, S. S.; Boey, Y. C. F.; Tan, L. P. Porous Polycaprolactone Scaffold for Cardiac Tissue Engineering Fabricated by Selective Laser Sintering. Acta Biomater. 2010, 6 (6), 2028e2034. 96. Wiria, F. E.; Leong, K. F.; Chua, C. K.; Liu, Y. Poly-ε-Caprolactone/Hydroxyapatite for Tissue Engineering Scaffold Fabrication via Selective Laser Sintering. Acta Biomater. 2007, 3 (1), 1e12. 97. Bartolomeu, F.; Buciumeanu, M.; Costa, M.; Alves, N.; Gasik, M.; Silva, F.; Miranda, G. MultiMaterial Ti6Al4V & PEEK Cellular Structures Produced by Selective Laser Melting and Hot Pressing: A Tribocorrosion Study Targeting Orthopedic Applications. J. Mech. Behav. Biomed. Mater. 2019, 89, 54e64. 98. Costa, M.; Bartolomeu, F.; Palmeiro, J.; Guimar~aes, B.; Alves, N.; Miranda, G.; Silva, F. MultiMaterial NiTi-PEEK Hybrid Cellular Structures by Selective Laser Melting and Hot Pressing: Tribological Characterization. Tribol. Int. 2021, 156, 106830. 99. Ziaee, M.; Crane, N. B. Binder Jetting: A Review of Process, Materials, and Methods. Addit. Manuf. 2019, 28, 781e801. 100. Mostafaei, A.; Elliott, A. M.; Barnes, J. E.; Li, F.; Tan, W.; Cramer, C. L.; Nandwana, P.; Chmielus, M. Binder Jet 3D PrintingdProcess Parameters, Materials, Properties, Modeling, and Challenges. Prog. Mater. Sci. 2021, 119, 100707. 101. Thakar, C.; Deshmukh, S. P.; Mulla, T. A Review on Selective Deposition Lamination 3D Printing Technique. Int. J. Adv. Sci. Res. Eng. Trends 2020, 4, 7e11. 102. Zhou, W. Y.; Lee, S.; Wang, M.; Cheung, W. Selective Laser Sintering of Tissue Engineering Scaffolds Using Poly (L-Lactide) Microspheres. In Key Engineering Materials; Trans Tech Publ, 2007. 103. Higa, C. F.; Gradowski, T.; Elifio-Esposito, S.; et al. Influence of Selective Laser Sintering Process Parameters on Microstructure and Physicochemical Properties of Poly (Vinyl Alcohol) for the Production of Scaffolds. Rapid Prototyp. J. 2020, 26 (6), 1155e1164. 104. Chen, P.; Cai, H.; Li, Z.; Li, M.; Wu, H.; Su, J.; Wen, S.; Zhou, Y.; Liu, J.; Wang, C. Crystallization Kinetics of Polyetheretherketone During High Temperature-Selective Laser Sintering. Addit. Manuf. 2020, 36, 101615. 105. Jonkers, N.; van Dijk, W. J.; Vonk, N. H.; van Dommelen, J. A. W.; Geers, M. G. D. Anisotropic Mechanical Properties of Selective Laser Sintered Starch-Based Food. J. Food Eng. 2022, 318, 110890. 106. Wu, B. M.; Borland, S. W.; Giordano, R. A.; Cima, L. G.; Sachs, E. M.; Cima, M. J. Solid Free-Form Fabrication of Drug Delivery Devices. J. Contr. Release 1996, 40 (1e2), 77e87. 107. Cheng, T.-Y.; Liao, Y.-C. Rapid Hardening Process for Starch-Based Powder Bed 3D Printing. J. Manuf. Process. 2022, 75, 259e267. 108. Holland, S.; Tuck, C.; Foster, T. Selective Recrystallization of Cellulose Composite Powders and Microstructure Creation Through 3D Binder Jetting. Carbohydr. Polym. 2018, 200, 229e238. 109. El Awad Azrak, S. M.; Clarkson, C. M.; Moon, R. J.; Schueneman, G. T.; Youngblood, J. P. Wet-Stacking Lamination of Multilayer Mechanically Fibrillated Cellulose Nanofibril (CNF) Sheets with Increased Mechanical Performance for Use in High-Strength and Lightweight Structural and Packaging Applications. ACS Appl. Polym. Mater. 2019, 1 (9), 2525e2534. 110. Kang, D. H.; Louis, F.; Liu, H.; Shimoda, H.; Nishiyama, Y.; Nozawa, H.; Kakitani, M.; Takagi, D.; Kasa, D.; Nagamori, E. Engineered Whole Cut Meat-Like Tissue by the Assembly of Cell Fibers Using Tendon-Gel Integrated Bioprinting. Nat. Commun. 2021, 12 (1), 1e12. 111. Mehrotra, S.; Kumar, S.; Srivastava, V.; Mishra, T.; Mishra, B. N. 3D Bioprinting in Plant Science: An Interdisciplinary Approach. Trends Plant Sci. 2020, 25 (1), 9e13.

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112. Krujatz, F.; Dani, S.; Windisch, J.; et al. Think Outside the Box: 3D Bioprinting Concepts for Biotechnological ApplicationseRecent Developments and Future Perspectives. Biotechnol. Adv. 2022, 58, 107930. 113. Cubo-Mateo, N.; Podhajsky, S.; Knickmann, D.; Slenzka, K.; Ghidini, T.; Gelinsky, M. Can 3D Bioprinting be a Key for Exploratory Missions and Human Settlements on the Moon and Mars? Biofabrication 2020, 12 (4), 043001. 114. Liu, F.; Vyas, C.; Yang, J.; Ates, G.; B
artolo, P. J. A Review of Hybrid Biomanufacturing Systems Applied in Tissue Regeneration; Virtual Prototyping & Bio Manufacturing in Medical Applications, 2021; pp 187e213.

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CHAPTER 3

Biopolymers in additive manufacturing Ana C. Lemos de Moraisa, b, Vincent S.D. Voeta, Rudy Folkersmaa and Katja Loosb a

Professorship Sustainable Polymers, NHL Stenden University of Applied Sciences, Emmen, the Netherlands; Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands b

1. Introduction Additive manufacturing (AM) is one of the most widespread processes used to rapidly prototype and directly manufacture custom plastic parts from computer-based design models (1). Different materials for 3D printing exist or are being created for different types of applications. Polymers like poly(lactic acid) (PLA) and acrylonitrile butadiene styrene (ABS) have been utilized extensively. The global market for AM materials revenues is expected to increase to more than $30 billion in the next decade (2). As a result, there will be a huge demand for new materials for 3D printing that will replace current plastics with more eco-friendly and sustainable alternatives. The investigation of renewable and natural polymers for the manufacturing of different 3D products has generated widespread attention in academia and industry (3). In recent decades, biodegradable and biobased polymers have garnered considerable interest, because of the environmental concerns with regard to the plastic soup and large carbon footprint of fossil-based materials. Biopolymers have been recognized by the European Union (EU) as a sustainable alternative to synthetic plastics in relation to finite fossil resources, a healthy environment, and climate change. Currently, the production and application of bioplastics have been progressively explored by research communities where the main focus of research has been on technological aspects, environmental issues, and sustainability effects. In certain cases, the application of biopolymers is unavoidable due to their favorable biocompatibility, biodegradability, as well as non-toxicity (4,5). Biopolymers are extensively available and they can be possibly used in diverse applications including paper coating, packaging, food science, biomedical systems, and agriculture. Biopolymers can be derived in two different ways; the first consists of renewable sources (microorganisms, animals, plants, and also proteins). Polyhydroxyalkanoates (PHAs), polysaccharides (starch, cellulose, alginate, pectin chitosan), and PLA are some considerable examples. Another type of biopolymer is derived from petroleum. Poly(butylene succinate) (PBS), polycaprolactone (PCL), and certain aliphatic-aromatic copolyesters, namely poly(butylene succinate-co-terephthalate) (PBST) and poly(butylene adipate-co-terephthalate) (PBAT) should be stated because they are already being used in industrial applications (6).

Additive Manufacturing of Biopolymers ISBN 978-0-323-95151-7, https://doi.org/10.1016/B978-0-323-95151-7.00001-6

© 2023 Elsevier Inc. All rights reserved.

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The research of AM biopolymer materials has primarily focused on biomedical and food applications, though a broader range of applications, including wastewater treatment and sense, has also been reported (7). This chapter will discuss the main biopolymers that are used in additive manufacturing, schematically presented in Fig. 3.1, and explore their unique properties and applications.

2. Poly(lactic acid) (PLA) PLA is the most promising biopolymer used in AM, as compared to other biopolymers. The monomer, lactic acid (LA), can be obtained from sugar fermentation, which can be polymerized subsequently into a low-molecular-weight PLA pre-polymer. A catalytic reaction helps to convert the prepolymer into the cyclic dimer, lactide, which is then purified and polymerized into PLA by ring opening polymerization (4). PLA has good mechanical properties comparable to conventional fossil-based polymers. It is a semi-crystalline and amorphous solid and has a glass transition temperature (Tg) of about 55 C and a melting temperature of ca. 180 C depending on the exact chain structure (8). Because of the processing ease of PLA and its desired thermal properties, PLA is commonly utilized in AM, generally in the form of commercial monofilaments for fused deposition modeling (FDM) (9). A recent study showed the design and 3D printing of a sorbent cylinder using PLA with carbon black, a commercially available filament, for the elimination of volatile organic compounds (VOCs) (i.e., toluene, ethylbenzene, and

Fig. 3.1 Overview of biopolymers involved in additive manufacturing, discussed in this chapter.

Biopolymers in additive manufacturing

Fig. 3.2 Screen captures of infill layers of sorbent cylinders at fill densities. From left to right: 15%, 20%, 25%, 30%, 40%, and 50%. (Reproduced with permission (10). Copyright 2019, Elsevier.)

benzene) from water (10). Individual sorbent cylinders at infill densities of 15%, 20%, 25%, 30%, 40%, and 50% were printed (Fig. 3.2) and tested in an aqueous solution containing the VOCs mentioned. The authors reported that, at the end of the 5h test, cylinders with the highest filling rate (50%) demonstrated the best result, removing 52% of benzene, 82% of toluene, and 92% of ethylbenzene from the aqueous solution (10). The development of intelligent materials for additive manufacturing technologies makes it possible to acquire new 3D printable products. Polymers that have shape memory effect (SME) present an additional dimension to the 3D printing known as 4D printing. These materials can recover to their original size and shape upon specific stimuli such as temperature change (11). Serjouei et al. (12) investigated the effect of design and process parameters of 4D printed sandwich structures on their stress absorption degree and shape recovery performance. They produced two distinct auxetic sandwich structures using PLA filaments via an FDM 3D printer and evaluated the stress absorption rate (Fig. 3.3). The experimental results revealed that the energy absorption rate is direct proportional to the internal cell count; nevertheless, the maximum deformation is significantly reduced compared with standard samples. The authors concluded that finding the optimal processing parameters is vital to prevent any changes in the maximum deformation depth of the sandwich structure (12).

Fig. 3.3 4D printed shape memory sandwich structures with hexagonal horseshoe shape of internal cells. (Reproduced under the terms of the Creative Commons CC BY license (12). Copyright 2022, IOP Science.)

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Fig. 3.4 Structural formula of poly(lactide-co-glycolide) (PLGA) diacrylates (top) and SEM image of a 3D printed lattice structure based on 75:25 PLGA-diacrylate (bottom) (13). (Copyright 2020, Elsevier.)

While PLA is well-known for its application in FDM, it has been applied in other 3D printing techniques as well. Wilts et al. (13) prepared a photoactive oligomeric precursor, poly(lactide-co-glycolide) (PLGA) diacrylate, via ring opening polymerization for application as liquid photoresin in vat photopolymerization (VP). Six formulations of PLGA oligomers with varying molar ratios were synthesized. The crosslinked networks revealed elastic moduli in the range 0.01e0.03 MPa and Tg’s oscillating from 17 to 20 C, demonstrating their potential application in soft tissue scaffolds. Tests for cytotoxicity and cell attachment presented a reduction of the cell viability for lower acrylate conversions, related to the presence of free (cytotoxic) acrylate. PLGA 75:25 (L:G) was successfully VP printed on a stereolithography apparatus (SLA), and the fabricated microlattice demonstrated high feature resolution and excellent surface finish (Fig. 3.4) (13).

3. Polycaprolactone (PCL) PCL is a biodegradable and biocompatible aliphatic polyester, with an indispensable polymer matrix for tissue support because of its properties that include gradual resorption after implantation, non-toxicity, and adequate mechanical properties. PCL has a melting point below 100 C, making it a valuable matrix material for biofillers, as most (bio)polymers do not degrade in the PCL polymer processing window. This affords the chance to combine PCL with new filler materials, such as reinforcing agents, to enhance matrix properties (14).

Biopolymers in additive manufacturing

PCL is hydrophobic, so its degradation time is larger than PLA, making it suitable for applications that require long degradation times. PCL is reabsorbed by hydrolysis of the ester bonds in the human body. As a result, it has gained considerable interest as a material for implantable medical devices, similar to PLA. PCL has been formulated with various pharmaceuticals for the delivery of drugs and specific targeted release (15). PCL scaffolds were also developed for bone and cartilage tissue engineering, due to PCL’s biocompatibility, slow degradation rate, less acidic by-products during the degradation compared to other polyesters, and its potential for loadbearing applications (16,17). A study used PCL melt blended with 20% (mass fraction) nano- and microhydroxyapatite (HA) to produce composite tissue engineering scaffolds created by a self-developed FDM 3D printer (18). The results demonstrated that the inclusion of HA rises the PCL’s crystallization rate as well as crystallization ability. Both composites, with micro- and nano-HA, showed the presence of pores that promote cell adhesion, nutrient transport, and proliferation. However, because of the aggregation of HA particles in the micro-HA/PCL scaffold for tissue engineering, the tensile and flexural strengths of nano-HA/PCL increase. The authors conclude that the nano-HA/PCL composite has the largest potential for bone tissue engineering (18). Photopolymerizable resins based on PCL copolymers were synthesized and applied in digital light processing (DLP) to produce bioresorbable tissue engineering implants (19). First, caprolactone was copolymerized with trimethylenecarbonate (TMC) and subsequently functionalized with urethane acrylate. By controlling the copolymer ratio, the mechanical properties and degradation rate of 3D printed constructs were tailored.

4. Polyhydroxyalkanoates (PHAs) In recent years, research on biopolymers derived from fermentation driven by microorganisms has accelerated. Similar to PLA, PHAs are an extensive group of polyesters. However, their lower Tg especially in comparison to PLA causes them to exhibit different physical properties (4). Due to its crystallinity, polyhydroxybutyrate (PHB) is indeed a sustainable replacement for current AM materials among the existing PHAs (Fig. 3.5). However, it has a

Fig. 3.5 Structural formula and nomenclature of polyhydroxyalkanoates (PHAs). (Reproduced under the terms of the Creative Commons CC BY license (4). Copyright 2021, Elsevier.)

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limited processing range of temperature and is extremely brittle. On the other hand, poly(3-hydroxybutyrate-co-hydroxy valerate) (PHBV), a copolymer of PHB along with hydroxyvalerate (HV), demonstrates a broader processing window, lower melting point, lower crystallinity, heat resistance, and good mechanical strength. However, PHBV remains susceptible to thermal decomposition and is far more ductile than PHB, restricting its practical applications (12). 4.1 Polyhydroxybutyrate (PHB) A case study related to PHB in additive manufacturing was developed by Saska et al. (20). In this research, structural characterization and in vitro analysis were carried out on PHB scaffolds that are manufactured by selective laser sintering (SLS), following the adsorption of osteogenic growth peptide (OGP). The morphological analysis showed that SLS printing enabled the creation of an intrinsic porosity (Fig. 3.6) that is conducive to cell adhesion and proliferation. In addition, the morphology of the rods was very well-kept, and the pores can be recognized in comparison to the software model. Because compressive strength is essential for bone tissue scaffolds and must ideally fit those of living bone, it was carefully evaluated. PHB scaffolds had values below 1 Mpa. Despite the fact that the compressive strength of PHB scaffolds was significantly lower than that of human cancellous bone (10 MPa), these values were greater than those taken from other SLS-printed scaffolds. The controlled release of the labeled OGP peptide from the PHB scaffolds indicated, according to the authors, that the fabricated scaffolds have the potential to serve as drug delivery matrices (20).

Fig. 3.6 (A) PHB scaffold 3D printed via selective laser sintering. (B) Optical micrograph presenting pore structure in scaffolds. (Reproduced with permission (20). Copyright 2018, Elsevier.)

Biopolymers in additive manufacturing

4.2 Poly(3-hydroxybutyrate-co-hydroxy valerate) (PHBV) PHBV has difficulties to support proliferation, cell adhesion, as well as differentiation. Furthermore, the low mechanical properties of PHBV restrict its advance in clinical application, particularly in bone tissue engineering. Calcium sulfate hemihydrate (CaSH) is osteoconductive and biocompatible and has already been applied clinically in bone repair and prosthetic alternative materials. CaSH could therefore increase the rigidness and hydrophilicity of polymeric scaffolds (21). In a study involving 3D printing of PHBV/CaSH composite scaffolds, this material shows the potential for bone repair (21). PHBV was mixed with 20% pure CaSH via melt blending using a twin-screw extruder. The standard PHBV/CaSH composite scaffold was 3D printed by an FDM system (Fig. 3.7). Printed PHBV/CaSH scaffolds were impregnated in 3 wt.% chitosan acetic acid solution (CS), to further promote cell adhesion and proliferation. Compared to the PHBV and PHBV/CaSH scaffolds, PHBV/CaSH/CS scaffolds promoted the proliferation and adhesion of rat bone marrow stromal cells (rBMSCs)

Fig. 3.7 Photographs and SEM images of the various PHBV-based scaffolds produced. (Reproduced with permission (21). Copyright 2018, Elsevier.)

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and also enhanced the osteogenesis of rBMSCs by upregulating the expression level of osteogenic genes. In vivo studies revealed no bone formation in pure PHBV scaffolds. In case of the PHBV/CASH scaffolds, new bone formation was insignificant at 4 weeks, while new bone tissue was formed in small amounts after 12 weeks. On the other hand, the PHBV/CaSH/CS scaffolds already formed new bone tissue after 4 weeks of implantation. Thus, the authors concluded PHBV/CaSH/CS scaffolds induced more newly formed bone with respect to the other scaffolds produced (21).

5. Proteins Proteins are organic polymers composed of amino acid residues connected by peptide bonds. Keratin, gelatin, silk, and soy protein are utilized in a variety of fields due to their superior biocompatibility, nutritional, and functional qualities (7). The repetitive nature of proteins facilitates the creation of long-range ordered molecular secondary structures that are capable of self-assembly into complex three-dimensional hierarchical architectures with unique chemical, mechanical, electrical, optical, and electromagnetic properties. Due to their low thermal stability, protein-based AM relies primarily on the extrusion of aqueous solutions or suspensions. By utilizing inherent gelation properties or physicalchemical crosslinking, their physical stability in physiological environments is accomplished. In bioprinting techniques, the high cell affinity of proteins is primarily exploited (22). Due to their high molecular weight, organizational states, and supramolecular functionality, the proteins used in additive manufacturing typically undergo processing in their natural state. The adaptability of molecular geometry and the ease of mixing, aggregation, gelation, and deposition associated with printing applications enable proteins to create highly effective 3D printed structures. Protein printing research is in its infancy and focuses primarily on ink composition studies to achieve the required 3D structures (23). 5.1 Collagen Collagen, a structural protein found in abundance, is among the most commonly utilized biopolymers in 3D printing processes. As a structural protein, collagen is a vital component in connective tissue. Gel printing is possible following extrusion-based printing and enzymatic digestion (22). Collagen is a common biopolymer found in mammals and can be used in bone scaffolds or cardiovascular implants (24). Nevertheless, collagen in its natural state lacks the needed strength to form printed architectures, hence it is typically mixed with other biological materials. Due to the low consistency index and slow polymerization, maintaining functionality in the liquid state as a printable bioink is difficult. Collagen-based dispersions may be processed using inkjet, extrusion, and SLS printing techniques (23).

Biopolymers in additive manufacturing

Suo et al. (25) stated that chitosan and collagen composites are intriguing materials but generally show poor printability. In order to develop collagen/chitosan scaffolds with varying ratios, the researchers combined 3D printing at low temperatures with genipin cross-linking to improve the printability of the biopolymers through hydrogen bond interactions. The case study demonstrated that chitosan itself was not able to be printed at low temperatures, but collagen’s printability could be improved with chitosan inclusion. The authors believe that this happens due to the increased hydrogen bonding between chitosan and collagen. For the hybrid Col/Chi scaffolds, swelling tests proved that chitosan plays a leading role in the structure retainment, supposedly due to stronger molecular interactions. The concentration of collagen in the scaffolds is directly proportional to the degradation rate as a result of collagenase that can hydrolyze the three-dimensional spiral structure under physiological pH and temperature. The Col/Chi scaffold has a controllable pore structure, which offers space for the development and migration of cells and the transport of nutrients and metabolic waste (25). 5.2 Gelatin Gelatin is a fibrous protein derived from collagen’s partial hydrolysis. Gelatin can be extracted from bones and bovine hides, pigs, and fish skins. Gelatin has garnered a great deal of attention due to its distinctive attributes; it is thermo-reversible with a melting point that is close to body temperature, and it could also perform as a thickening and gelling agent that is simple to use. Based on conditions (temperature, pH, and concentration), gelatin has a broad variety of viscosity, allowing it to be processed via additive manufacturing (7,26). Gelatin is used as grafts in cardiology, as a bone substitute in orthopedics, and to create 3D biometrics in dermatology (24). Because of its biocompatibility, biodegradability, cross-linking properties, and thermal stability within the environment, it has been widely utilized for the production of functional printable inks (23). A recent case study proposed evaluating the printability of salmon skin gelatin gel at various concentrations by analyzing their physical properties (textural, rheological, and printing behavior) as well as dimensional stability (27). Physical properties (hardness, viscosity, gelling, and melting temperatures) and concentration of salmon gelatin were found to be significantly affected by extraction conditions. Salmon gelatin gels exhibit the ability to flow as a self-governing filament and form three-dimensional geometries. Analyzing Young’s modulus and extrusion hardness demonstrated that it was not possible to print with 2% salmon gelatin because printed objects did not retain their shape. In addition, the sample containing 14% salmon gelatin could not be extruded due to the extrusion force exceeding that of the printer. The sample containing 8% gelatin (Fig. 3.8) was optimal for application in the 3D printing process due to its superior printability, high dimensional stability, and shape retention (27). The authors proposed that the 3D printed matrices could be very beneficial to make customized food for individuals with specific needs in nutrition (27).

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Fig. 3.8 3D-printed (A) cube and (B) cylinder of 8% salmon gelatin gel. (Reproduced with permission (27). Copyright 2022, Elsevier.)

5.3 Soy protein Soy protein is a naturally occurring polypeptide composed primarily of globulins and albumins. It can be categorized as soy flour, soy protein concentrate (SPC), and soy protein isolate (SPI). SPI is the most refined type of soy protein with a low amount of protein content of 90%. It possesses superb properties, including heat-induced thermoplasticity, biocompatibility, and biodegradability, and has hence been utilized in a variety of areas (7). Soy protein is abundant in essential amino acids, possesses excellent functional and physicochemical attributes, and has been effectively printed to produce porous scaffolds that can be utilized as biomaterial implants for regenerative medicine applications (28). Beyond the biomedical field, soy protein has also been explored for food printing by Phuhongsung et al. (29). The authors investigated the effect of vanilla and carrageenan flavor on the geometrical shape quality and post-printing microwave irradiations of 3D-printed SPI-based foods and their flavor profiles. The compound with 3% of carrageenan and 0.5% of vanilla turned out to be suitable for 3D printing. The electronic tongue sensor verified the taste aspect of 3D printed products with and without vanilla flavor. SPI without vanilla flavor demonstrated that as temperature increases, characteristics of umami and saltiness intensify while the perceptions of sourness and bitterness keep unaffected among the specimens. Samples with vanilla flavor presented intensified taste for bitterness, astringency, umami, richness, and saltiness. According to the authors, this relates to the sensibility of vanilla flavor to temperature more than the real taste of SPI (29).

Biopolymers in additive manufacturing

5.4 PEA protein A recent study reported the use of pea protein in 3D printing. This protein has drawn attention for its application in the development of emulsions and gels, which is one of the most important functional properties of the globular proteins as it is used to influence food texture, expansion, processability, moisture-absorption, and moisture-retaining capacity (30,31). Oyinloye and Yoon (31) studied blends based on pea protein and alginate for application in 3D printing materials (Fig. 3.9), analyzing its rheology, thermal, and texture properties. Increasing the concentration of pea protein also increased the investigated blend properties, thereby affecting the extrusion behavior during 3D printing. The optimal blend for 3D printing was selected (80% alginate and 20% pea protein) and used for additive-layer manufacturing (ALM) simulation to investigate the required printing conditions (31). ALM simulation is a novel approach to additive manufacturing, applied to predict mechanical properties like stress and deformation during printing. In comparison to standard 3D printing, ALM simulation benefits the precision of material deposition and geometry flexibility, reducing waste generation and production costs (31). The researchers showed that the use of an additive simulation method for gel printing offers an opportunity to produce parts with more complex geometric architectures with predefined thermochemical characteristics. 5.5 Zein Zein is an alcohol-soluble prolamine storage protein found in corn that is rich in neutral amino acid and hydrophobic residues, as well as a few polar amino acid residues. It could be used as a hydrophobic printable ink for different AM applications such as bioengineering, food, and drug delivery systems. Zein is an expensive type of protein to obtain, and therefore, additive manufacturing would provide a significant economic benefit since it reduces the amount of waste material generated during production. Reports on the 3D printing of zein-based substances have been scarce, due to the brittleness and low flexibility of printed objects based on this protein (23,32).

Fig. 3.9 Additive-layer manufacturing of (A) alginate gel 100%, (B) AP 90:10, (C) AP 80:20, (D) AP 70:30. (Reproduced with permission (31). Copyright 2020, Elsevier.)

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Electrohydrodynamic printing (EHDP) of PCL/zein composite inks, however, was utilized by Jing et al. (33) to fabricate scaffolds for biomedical applications that were custom-made. Yield stress and Young’s modulus significantly increased the mechanical stability of scaffolds printed with PCL/zein composite inks. Enzyme-accelerated in vitro degradation indicates that the degradation rate of zein-containing scaffolds increased proportionally to the dose. When examined on mouse embryonic fibroblast and human lung cancer cells, PCL/zein scaffolds demonstrated superior cell affinity. Consequently, it shows the potential use of this mixture in 3D cell culture modeling, drug delivery systems, and tissue engineering applications (33). 5.6 Silk protein Silk is a natural protein fiber derived from arthropods like spiders and silkworms. Silk fibroin (SF) is compromised of around two-thirds crystalline and one-third amorphous conformations in one heavy chain of 390 kDa and one light chain of 25 kDa, joined by a disulfide link. SF is poorly soluble in diluted acid, water, and organic solvents (7). Being a versatile biopolymer, silk is known for its application in biomedical and tissue engineering and regenerative medicines including drug delivery and implantable devices, owing to its biocompatibility and wide range of physicochemical properties (34). Shin and Hyun (35) reported a 3D structuring of silk fibroin microneedles (Fig. 3.10) with riboflavin, an enzyme that initiates as a photoinitiator, using DLP 3D printing. After compression, no breakage or loss of sharpness was noted in the microneedles, confirming their ability to penetrate the skin. A flexible silk fibroin microneedle matrix pad was fabricated and subsequently used to deliver fluorescence dye molecules into pig skin to simulate drug delivery. It was found that larger needles would be required for the skin sample. The authors claimed that the developed generic one-step 3D constructing method can be applied for the manufacturing of protein-based microneedles in the near future (35). 5.7 Keratin Keratin is sourced from hair, wool, and feathers and could be applied in products of personal care and also wound dressings. Keratin consists of a central helical rod domain and variable N- and C-terminal domains. The protein could be processed in aqueous media and deposited as film or fibers by itself or in combined effect with synthetic or other biopolymers (36). The biopolymer is broadly utilized in skin, muscles, bone, and nerve reformation due to its excellent biodegradability, biocompatibility, and extremely low immune response following implantation (7). A recent study considered using keratin from chicken feathers and human hair as a reinforcement for PLA in additive manufacturing of polymeric scaffolds. Keratin was milled and mixed with PLA and chitosan by extrusion and the blends were 3D printed (Fig. 3.11) (37). Both the particle size and concentration of keratin within the composites

Biopolymers in additive manufacturing

Fig. 3.10 (A) Image of the microneedle array, (B) view of a single microneedle, (C) flexible pad with microneedles. (Reproduced with permission (35). Copyright 2021, Elsevier.)

Fig. 3.11 Process of 3D printing scaffolds of PLA and chitosan reinforced with keratin. (Reproduced with permission (37). Copyright 2020, Elsevier.)

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play a critical role in dispersion and also increase the compatibility between the components, generating more biocompatible materials verified by the excellent growth of cells. Also, composites with particles indicate a moderate swelling rate after 96 h in water and simulated body fluid (SBF), demonstrating sensitivity to contact with liquids. The authors concluded that the biopolymer scaffolds printed in this study show an important substitute for tissue engineering, for bone reformation (37). 5.8 Casein and whey protein The protein casein is a member of a family of linked phosphoproteins with supramolecular structures that ensure physical stability in dairy. In combination with hydrocolloids, casein generates a consistent heterogeneous network structure. Due to their sensorial, textural, and nutritional qualities, they are frequently used in the process of 3D printing (7,23). Additionally, whey protein is extracted from milk as well. It contains numerous essential amino acids and trace elements, including potassium, calcium, iron, and magnesium. It is frequently used in food production and processing. After being heated, gelatinous whey protein will denature and aggregate to form a three-dimensional network that can envelop other substances. Due to its ability to form hydrogels and its printability, whey protein is also widely utilized as a functional ink for 3D printing (23,38). Daffner et al. (39) have studied casein and whey protein together as suspensions and investigated their usage for extrusion-based 3D printing for personalized nutrition. The influence of different pH values during heat treatment was investigated, as was the use of acidification to control the aggregation rate and sol-gel transition temperature of caseinwhey protein suspensions. The aggregation rate was increased by lower pH levels and higher protein concentrations. At pH values of 4.8 and 5.0, extrusion-based 3D printing produced gels that were firm and stable, with more adequate formulations discovered at higher temperatures (39).

6. Polysaccharides Polysaccharides, sometimes referred to as carbohydrate polymers, are natural or synthetic macromolecules obtained through polymerization of one or more monosaccharides via glycosidic covalent bonds. Due to their low costs, renewability, and widespread availability, polysaccharides including starch, alginate, cellulose, chitosan, carrageenan, hyaluronic acid and pectin have been applied in food, pharmaceuticals, biomedicals, electronics, advertising, etc. These polysaccharides can be obtained from a variety of natural sources and have complex semicrystalline structures resulting in strong intermolecular as well as intramolecular hydrogen bonds. Properties such as biodegradability and mechanical performance depend largely on their architecture and crystallinity (7,40).

Biopolymers in additive manufacturing

6.1 Cellulose Cellulose is one of the utmost rich biodegradable and sustainable biopolymers in the world. It is a homogeneous polysaccharide made up of linear b-(1 / 4)-glucan with intra- and intermolecular hydrogel networks (41). Due to their exceptional mechanical attributes, cellulose fibers have been utilized to reinforce biomaterial matrices used in extrusion-based 3D printing. Similarly, nanocellulose (in the form of cellulose nanocrystals (CNCs) or cellulose nanofibrils (CNFs)) has been utilized as reinforcing agents in inks intended for AM printing (42). Until now, cellulose and its derivative products have emerged as biomaterials with good potential for 3D printing and also have provided a feasible means of fabricating delicate and sustainable structures (3). A recent study involving this polysaccharide employed CNCs as a renewable and safe rheology modifier, which is capable of enabling 3D direct ink writing (DIW) printing of puree spinach, applesauce, and tomato puree, and using lyophilization to get solid structures (43). Inks of printable CNC-food comprise 5 or 7.5 vol% freeze-dried CNCs integrated with a puree of food that is bought at a neighborhood supermarket with over 88% of water content. By using spinach, tomato, and applesauce-based inks, the research team printed octopi (Fig. 3.12A and B) and pyramid models (Fig. 3.12C and D). Regardless of differences in food base composition, three consistent shifts in rheological behavior had been observed: the maximum viscosity raised linearly as the concentration of CNC was increased, the edible inks created exhibited thinning behavior, and they demonstrated primarily elastic, gel-like performance in the linear viscoelastic elastic

Fig. 3.12 DIW printed food with 5% (A, C) and 7.5% (B, D) of CNC, using applesauce (left) spinach (middle) and tomato (right), as printed (top) and post-cooling (bottom). (Reproduced with permission (43). Copyright 2022, Elsevier.)

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region. Applesauce-based inks exhibited the best external finish after freeze-drying, while structures printed with inks derived from spinach and tomato exhibited voids and surface roughness. The outer layer of the CNC serves as a decomposable, edible protective shell, that has the ability to decrease or remove the packaging for edible printed structures. Results clearly indicate a promising future for CNCs in expanding the variety of nonnatively printable food, thereby allowing more applications of edible 3D printing (43). 6.2 Lignin Lignin, as one of the main components of black liquor from the process of chemical pulping, is one of the most abundant organic polymers in the world and also the only highvolume renewable raw material that is composed of aromatics. More than 50 million tons of lignin are manufactured each year, however, 98% is incinerated to generate energy and just 2% of lignin is used for other purposes, primarily in applications like fillers and dispersants. Nowadays, lignin is attracting more and more global attention and shows great economic potential for the 3D printing materials market, due to its biodegradability, low cost, as well as high (renewable) carbon content (3). A low-cost direct ink printing method for the fabrication of lignin-based 3D shapes was implemented (44). The rheological properties of ink containing lignin are changeable by a wetting agent, from soft to rigid, allowing vertical printing that needs rigid and self-supporting properties. Successful additive manufacturing of lignin through a simple and scalable DIW technology provides a promising route for fabricating thermally stable, mechanically sturdy, as well as biocompatible structures (44). Moreover, this study shows that lignin could be printed in combination with other materials to form a composite product for applications in the field of water treatment. This expands the functional options for 3D lignin-based composite materials that are inapplicable to FDM printing. As a biodegradable substance derived from wood, the lignin-based 3D structures developed by ink printing have the ability to substitute plastics in specific applications, such as biomedical engineering. This will encourage continuous and healthy growth toward sustainable development for future wood-based material engineering (44). Lignin-containing photopolymer resins were incorporated in a commercial stereolithography system as well (45). Lignin was discovered to be a plasticizer, improving the elongation at break while decreasing Young’s modulus (Fig. 3.13). Printed samples demonstrate high surface definition and good layer fusion, however, the strong coloration caused by lignin may limit its future application (45). 6.3 Starch The majority of starch is extracted from cereals, tubers, and roots of various origins, including corn, cassava, wheat, rice, and potato. It consists primarily of two

Biopolymers in additive manufacturing

Fig. 3.13 Lignin-based methacrylate resins were applied in vat photopolymerization (left). With increasing lignin content, elongation increased while stiffness decreased (right). Lignin acts as a plasticizer in the 3D printed samples. (Reproduced with permission (45). Copyright 2018, American Chemical Society.)

biomacromolecules: linear amylose with (1,4)-linked D-glucose units and highly branched amylopectin with (1,4)-linked D-glucose backbones and (1,6)-linked branches (7). In AM, starch is most commonly used as a thickening or gelling agent, or rheology modifier. The inclusion of starch in ink formulations enhances printability, providing the ink with shear-thinning characteristics and also maintaining the shape of the printed structure itself (41). Zhang et al. (46) studied starch digestibility by hot extrusion 3D printing (HE-3DP) as a novel method to obtain starch-based food personalization. In this current research, structural alterations and in vitro digestibility of potato, wheat, as well as rice starch gel substances at various concentrations were analyzed during printing. Potato, wheat, and rice starch paste with concentrations of 10%e30% (w/w) were made and subsequently placed at 65 C into the printer cartridge. The samples were lyophilized in a vacuum freeze dryer after being frozen overnight at 80 C (46). Increasing concentrations of starch gel material could effectively resist hydrothermal or shear treatment disruption of the ordered structure, and thus encouraged macromolecular chain re-arrangements. Intriguingly, the newly created ordered structure increased the amount of slowly digestible starch in the printed samples significantly. Starch concentration was regarded as an essential parameter in controlling the ordered structure of printed samples and regulating their anti-digestibility (46). Acrylated cyclodextrin, an oligosaccharide derived from starch, was incorporated in UV-curable resin formulations suitable for vat photopolymerization. Increasing the content of cyclodextrin led to a higher crosslink density within the polymer network, resulting in an enhanced Tg. The biobased inks were successfully fabricated by DLP 3D printing with high geometrical fidelity (47).

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6.4 Alginate Alginate is a natural polymer that can be extracted via alkaline treatment from brown seaweed (Phaeophyceae). Because of its low price, low toxicity, and biocompatibility, alginate is widely used in the biomedical industry. Thanks to the advancement of alginate hydrogels, that can perform slight gelation when divalent cations are added, 3D printing of alginate is now possible. The field of 3D printing of alginate hydrogel scaffolds with controlled pore structures for tissue engineering applications is rapidly expanding (9). Most of the prepared bioink formulations used in AM include alginate to improve shape retention and mechanical strength, having a fast-ionic crosslinking ability. Alginate could be combined with different other biopolymers to create novel oriented printed components with improved printability (23). Ilhan et al. (48) studied the development of sodium alginate (SA) scaffolds together with polyethylene glycol (PEG) loaded with the antibacterial extract of Satureja cuneifolia (SC) as a possible treatment for diabetic ulcers. SA/PEG scaffolds were made by the addition of various concentrations (1%, 3%, and 5%) of PEG to 9% SA, while SC was included in 0.5%, 1%, and 2%. Because of the SC antibacterial extract they contain, 3D-printed scaffolds had an exceptional effect as an antibacterial drug, particularly opposing gram-positive bacteria. The cytotoxicity test revealed that the rates of biocompatibility were within the acceptable range. The authors hypothesized that antimicrobial printed composite scaffolds are an encouraging material for wound dressing and tissue engineering applications (48). 6.5 Pectin Pectins are a very common type of polysaccharide as well as a secondary component of plant cell walls. Numerous sources of pectin, like apples, are abundant and readily form hydrogels when mixed with water (49). Pectins consist of a diverse set of acidic polysaccharides that contain D-galacturonic acid residues. They are capable of producing a polymeric structure that is co-extensive with crosslinking glycans and cellulose. It is important to combine pectin with different biocomposite substances for its printability, which has already been accepted and utilized in the wound dressing, cooking, and cell culture industries (23). Because of its biodegradability, biocompatibility, adjustable physicochemical properties, excellent gelling properties, and functional modification potential, pectin has already found widespread application in the pharmaceutical industries, food industries, as well as in tissue engineering and current efforts have been focused on optimizing pectin-based inks for AM (50). Lapomarda et al. (51) developed green and non-toxic biomaterials based on pectin that was cross-linked with (3-glycidyloxypropyl)trimethoxysilane (GPTMS). The effect of GPTMS on the porosity, water uptake, mechanical properties, and cytocompatibility

Biopolymers in additive manufacturing

was investigated on microporous pectin/GPTMS sponges produced by freeze-drying. In addition, the printability of these compounds was studied. Prototypes of a human ear and nose manufactured by 3D printing pectin/GPTMS are illustrated in Fig. 3.14. The tests performed demonstrated that the increase of GPTMS reduced the porosity and had no influence on the pore size of the pectin sponges. Also, the water uptake was not affected by the crosslinker. Mechanically, an increase of GPTMS enhanced the compression modulus and this could be related to the rise of crosslink density inside the pectin matrix due to the addition of GPTMS. The compounds with pectin and GPTMS are cytocompatible. GPTMS offers a great advantage to pectin’s printability, permitting the production of 3D-shaped (patient-specific) scaffolds without the use of any support material (51).

Fig. 3.14 Human ear and nose produced by a 3D bioprinter. Pictures of prototypes (A, B) after bioprinting, and (C, D) after freeze-drying. (Reproduced with permission (51). Copyright 2020, American Chemical Society.)

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6.6 Chitin and chitosan Chitin is the second most widely available polymer, following cellulose, and is obtained from crustacean shells most frequently (14). This molecule is a linear polymer of b-1,4linked N-acetylglucosamine and is found in a variety of animals, such as arthropods, mollusks, echinoderms, and even mushrooms. Chitin is highly stable and indissoluble in water and the majority of organic solvents due to its macromolecular structure and strong intramolecular interactions. This poor solubility makes industrial applications of chitin challenging. Chitin is (partially) deacetylated to increase its solubility, yielding chitosan (43). Chitosan, a biopolymer, and biodegradable in origin, is used for the repair and fabrication of natural compatible tissues. Biocompatible chitosan includes a large number of primary amines and acetamido moieties, generating powerful intermolecular and intramolecular forces, resulting in a higher crystallinity degree and more rigid mechanical performance (52). Gu et al. (53) studied the use of chitin nanocrystals (ChiNC) as rheological modifier assistant to poly(1,8-octanediol-co-Pluronic F127 citrate) (POFC) to make pickeringlike emulsion inks and explore their printability through the DIW process. ChiNC was also a supporting agent to restrain filament collapse during thermocuring and at the same time performed as a biobased nanofiller for reinforcement of printed scaffolds (Fig. 3.15). The authors concluded from rheological experiments that the pre-POFC/ChiNC composite inks at 40% (w/w) content of solid satisfy the necessities for the DIW process. Subsequent thermo-crosslinking of the printed structures resulted in the ChiNC reinforced nanocomposite elastomer scaffolds. The printed scaffolds demonstrated low swelling and high potency and resiliency. The whole procedure was simple and ecological, requiring no toxic substances (53). A case study by Saatcioglu et al. (54) led to three-dimensional tissue scaffolds using a 3D printing procedure with chitosan as the polymer matrix and Osage orange extract (OGE) added in varying amounts as bioactive material. The findings indicate that the

Fig. 3.15 Photographs of deposited filaments extruded from pre-POFC/ChiNC-composite inks. (Reproduced with permission (53). Copyright 2020, Elsevier.)

Biopolymers in additive manufacturing

addition of OGE maximized the pore size of the scaffolds, which are non-toxic and biocompatible. The mechanical analyses demonstrated that with the addition of OGE, tensile strength declined proportionally. The swellability of chitosan was reduced with the addition of OGE. The manufactured scaffolds did not show antibacterial activity opposing S. aureus and E. faecalis (54).

7. Vegetable oils Vegetable oils are among the most accessible renewable resources, and their use in the production of environmentally responsible and sustainable materials is gaining popularity. The relatively low molecular weight of vegetable oils makes them appropriate building blocks for a variety of biobased polymers, but their low chemical reactivity makes their application challenging. Recent developments in technologies and methods for altering the chemical structure of vegetable oils have made them commercially available and economically feasible for a variety of industries, like coatings and inks industries (55). Oils derived from plants are one of the most frequently cited sources for bio-based resins. The double bonds in fatty acids are chemically modified to allow photocuring in stereolithographic printers to form a polymeric network (56). Epoxidized soybean oil (ESO) can be applied directly as resin in vat photopolymerization (57), although in most studies the acrylated form is used to produce printed objects of higher quality (58). Recently, a dual-curing system was developed, in which hybrid resins composed of both acrylates and epoxides were polymerized into interpenetrating networks during SLA printing (59). Guit et al. compared photoresins based on epoxidized soybean oil acrylate (ESOA) and epoxidized soybean oil methacrylate (ESOMA), prepared through a solvent-free synthesis pathway. Resins with up to 80% bio-renewable carbon were chosen for DLP printing, and manufactured prototypes showed a high feature resolution and excellent layer fusion (Fig. 3.16). Incorporation of ESOMA in the resins enhanced strength and stiffness with respect to ESOA, and the mechanical performance could be tuned further by changing the methacrylate functionality (60). The disposal of used vegetable oil for cooking is an environmental concern, given that global production is growing rapidly. In a recent study, waste cooking oil (WCO) from fast food restaurants was acrylated and successfully 3D printed in the presence of a photoinitiator, enabling a new route toward recycling WCO via AM (61).

8. Conclusions and outlook AM technology provides ample opportunities for on-demand and custom-made fabrication of (bio)medical, and consumer products. Considering the fast-growing market, it is essential to develop AM technology that accommodates responsible consumption and

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Fig. 3.16 Vat photopolymerization of biobased methacrylate photoresins derived from soy oil: (AeC) Pictures of 3D printed rook tower prototypes from various resin formulations. (D) Picture of 3D printed university logo. (E) SEM image of the internal helix structure in the rook tower prototype. (Reproduced with permission (60). Copyright 2020, American Chemical Society.)

production of goods while protecting Earth’s biosphere. AM materials, such as filaments, inks, and resins for 3D printing, play an important role in defining sustainability and therefore have to rely on eco-friendly renewable resources instead of fossil-based raw materials. Biobased and biodegradable polymers can be obtained from plants, animals, or microbes. Polylactide, polycaprolactone, and polyhydroxyalkanoate have been applied directly in material extrusion and powder bed fusion 3D printing. Their biocompatible, non-toxic properties make them ideal candidates for biomedical applications such as tissue engineering and bone regeneration. Incorporation of PLA or PCL oligomers to ink formulations for vat photopolymerization enables (bio)degradation in certain conditions, but typically requires chemical modification of functional (end) groups first, to allow photocrosslinking. Extrusion-based printing with naturally occurring proteins (e.g., gelatin, casein, soy protein), and polysaccharides like cellulose and starch is often studied for the production and processing of (personalized) food. These biopolymers can influence food texture, processability, and moisture absorption, and may even control digestibility. A more

Biopolymers in additive manufacturing

wide-scale application of proteins and polysaccharides in AM technology is still prevented, due to the inferior mechanical performance of the printed objects. Altering the chemical characteristics, however, may improve layer adhesion. Vegetable oils turn out to be valuable biobased building blocks for photocurable resins and have been extensively studied in stereolithographic 3D printing. The use of biopolymers in AM contributes to the reduction of the ecological footprint. The latest developments discussed in this chapter will inspire future researchers to explore the usage of other biopolymers, such as poly(butylene succinate) and poly(glycolic acid), in 3D printing methods. Moreover, circular end-of-life solutions should be pushed, such as recycling and (bio)degradation of printed parts, to reduce plastic waste. Biopolymers play an important role in facilitating this sustainable development. To further limit the effects of AM on the environment, it is imperative to pursue a closed-loop production cycle. To quantify the potential environmental impact of 3D printing processes and (bio)materials, life-cycle assessments must be performed standardly. Since AM is a highly multidisciplinary field, a broader collaboration between materials scientists (polymer), chemists, mechanical engineers, computer scientists, and adjacent disciplines is required to provide sustainable, biobased, and fully circular alternatives.

References [1] Chaunier, L.; et al. Material Extrusion of Plant Biopolymers: Opportunities & Challenges for 3D Printing. Addit. Manuf. 2018, 21 (March), 220e233. [2] Jafferson, J. M.; Chatterjee, D. Materials Today: Proceedings A Review on Polymeric Materials in Additive Manufacturing. Mater. Today Proc. 2021, 46, 1349e1365. [3] Yang, J.; et al. Cellulose, Hemicellulose, Lignin, and Their Derivatives as Multi-Components of BioBased Feedstocks for 3D Printing. Carbohydr. Polym. 2020, 250 (29). [4] Mehrpouya, M.; et al. Additive Manufacturing of Polyhydroxyalkanoates (PHAs) Biopolymers: Materials, Printing Techniques, and Applications. Mater. Sci. Eng. C 2021, 127 (April). [5] Shevchenko, T.; et al. Promising Developments in Bio-Based Products as Alternatives to Conventional Plastics to Enable Circular Economy in Ukraine. Recycling 2022, 7 (20). [6] Moeini, A.; et al. Formulation of Secondary Compounds as Additives of Biopolymer-Based Food Packaging: A Review. Trends Food Sci. Technol. 2021, 114 (May), 342e354. [7] Li, N.; et al. 3D Printing to Innovate Biopolymer Materials for Demanding Applications: A Review. Mater. Today Chem. 2021, 20. [8] Bhatia, A.; Sehgal, A. K. Additive Manufacturing Materials, Methods and Applications: A Review. Mater. Today Proc. 2021. [9] Fijoł, N.; Aguilar-S
anchez, A.; Mathew, A. P. 3D-Printable Biopolymer-Based Materials for Water Treatment: A Review. Chem. Eng. J. 2022, 430 (August 2021). [10] Lagalante, L. A.; Lagalante, A. J.; Lagalante, A. F. 3D Printed Solid-Phase Extraction Sorbents for Removal of Volatile Organic Compounds from Water. J. Water Proc. Eng. 2020, 35 (October 2019), 101194. [11] Barletta, M.; Gisario, A.; Mehrpouya, M. 4D Printing of Shape Memory Polylactic Acid (PLA) Components: Investigating the Role of the Operational Parameters in Fused Deposition Modelling (FDM). J. Manuf. Process. 2021, 61, 473e480. [12] Serjouei, A.; et al. 4D Printed Shape Memory Sandwich Structures: Experimental Analysis and Numerical Modeling. Smart Mater. Struct. 2022, 31.

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[13] Wilts, E. M.; et al. Vat Photopolymerization of Liquid, Biodegradable PLGA-Based Oligomers as Tissue Scaffolds. Eur. Polym. J. 2020, 130 (February), 109693. [14] Karimipour-Fard, P.; et al. Development, Processing and Characterization of Polycaprolactone/ Nano-Hydroxyapatite/Chitin-Nano-Whisker Nanocomposite Filaments for Additive Manufacturing of Bone Tissue Scaffolds. J. Mech. Behav. Biomed. Mater. 2021, 120 (May), 104583. [15] Chen, J. M.; et al. Solution Extrusion Additive Manufacturing of Biodegradable Polycaprolactone. Appl. Sci. 2020, 10 (9). [16] Yang, X.; et al. The Application of Polycaprolactone in Three-Dimensional Printing Scaffolds for Bone Tissue Engineering. Polymers 2021, 13 (16). [17] Wei, P.; et al. IGF-1-Releasing PLGA Nanoparticles Modified 3D Printed PCL Scaffolds for Cartilage Tissue Engineering. Drug Deliv. 2020, 27 (1), 1106e1114. [18] Jiao, Z.; et al. 3D Printing of HA/PCL Composite Tissue Engineering Scaffolds. Adv. Ind. Eng. Polym. Res. 2019, 2 (4), 196e202. [19] Kuhnt, T.; et al. Poly(Caprolactone-co-Trimethylenecarbonate) Urethane Acrylate Resins for Digital Light Processing of Bioresorbable Tissue Engineering Implants. Biomater. Sci. 2019, 7 (12), 4829e5522. [20] Saska, S.; et al. Three-Dimensional Printing and In Vitro Evaluation of Poly(3-Hydroxybutyrate) Scaffolds Functionalized with Osteogenic Growth Peptide for Tissue Engineering. Mater. Sci. Eng. C 2018, 89 (March 2017), 265e273. [21] Ye, X.; et al. Integrating 3D-Printed PHBV/Calcium Sulfate Hemihydrate Scaffold and Chitosan Hydrogel for Enhanced Osteogenic Property. Carbohydr. Polym. 2018, 202, 106e114. [22] Puppi, D.; Chiellini, F. Biodegradable Polymers for Biomedical Additive Manufacturing. Appl. Mater. Today 2020, 20. [23] Shahbazi, M.; J€ager, H. Current Status in the Utilization of Biobased Polymers for 3D Printing Process: A Systematic Review of the Materials, Processes, and Challenges. ACS Appl. Bio Mater. 2021, 4 (1), 325e369. [24] Udayakumar, G. P.; et al. Biopolymers and Composites: Properties, Characterization and their Applications in Food, Medical and Pharmaceutical Industries. J. Environ. Chem. Eng. 2021, 9 (4), 105322. [25] Suo, H.; et al. Low-Temperature 3D Printing of Collagen and Chitosan Composite for Tissue Engineering. Mater. Sci. Eng. C 2021, 123 (September 2020), 111963. [26] Tang, C.; et al. Collagen and Its Derivates from Structure and Properties to Their Applications in Food Industry. Food Hydrocolloids 2022, 107748. [27] Carvajal-Mena, N.; et al. Valorization of Salmon Industry by-Products: Evaluation of Salmon Skin Gelatin as a Biomaterial Suitable for 3D Food Printing. LWT (Lebensm.-Wiss. Technol.) 2022, 155. [28] Chen, J.; et al. Application of Soy Protein Isolate and Hydrocolloids Based Mixtures as Promising Food Material in 3D Food Printing. J. Food Eng. 2019, 261 (December 2018), 76e86. [29] Phuhongsung, P.; Zhang, M.; Bhandari, B. 4D Printing of Products Based on Soy Protein Isolate via Microwave Heating for Flavor Development. Food Res. Int. 2020, 137 (May), 109605. [30] Lam, A. C. Y.; et al. Pea Protein Isolates: Structure, Extraction, and Functionality. Food Rev. Int. 2018, 34 (2), 126e147. [31] Oyinloye, T. M.; Yoon, W. B. Stability of 3D Printing Using a Mixture of Pea Protein and Alginate: Precision and Application of Additive Layer Manufacturing Simulation Approach for Stress Distribution. J. Food Eng. 2020, 288 (May 2020), 110127. [32] Rowat, S. J.; Legge, R. L.; Moresoli, C. Plant Protein in Material Extrusion 3D Printing: Formation, Plasticization, Prospects, and Challenges. J. Food Eng. 2021, 308 (December 2020), 110623. [33] Jing, L.; et al. Zein Increases the Cytoaffinity and Biodegradability of Scaffolds 3D-Printed with Zein and Poly(ε-caprolactone) Composite Ink. ACS Appl. Mater. Interfaces 2018, 10 (22), 18551e18559. [34] Chouhan, D.; Mandal, B. B. Silk Biomaterials in Wound Healing and Skin Regeneration Therapeutics: From Bench to Bedside. Acta Biomater. 2020, 103, 24e51. [35] Shin, D.; Hyun, J. Silk Fibroin Microneedles Fabricated by Digital Light Processing 3D Printing. J. Ind. Eng. Chem. 2021, 95, 126e133. [36] Grigsby, W. J.; et al. Combination and Processing Keratin with Lignin as Biocomposite Materials for Additive Manufacturing Technology. Acta Biomater. 2020, 104, 95e103.

Biopolymers in additive manufacturing

[37] Rojas-Martínez, L. E.; et al. 3D Printing of PLA Composites Scaffolds Reinforced with Keratin and Chitosan: Effect of Geometry and Structure. Eur. Polym. J. 2020, 141 (August), 110088. [38] Du, Y.; Zhang, M.; Chen, H. Effect of Whey Protein on the 3D Printing Performance of Konjac Hybrid Gel. LWT (Lebensm.-Wiss. Technol.) 2021, 140 (November 2020), 110716. [39] Daffner, K.; et al. Design and Characterization of CaseineWhey Protein Suspensions via the pHe Temperature-Route for Application in Extrusion-Based 3D-Printing. Food Hydrocolloids 2021, 112 (February 2020). [40] Seidi, F.; et al. Crystalline Polysaccharides: A Review. Carbohydr. Polym. 2022, 275 (September 2021), 118624. [41] Liu, J.; et al. Current Advances and Future Perspectives of 3D Printing Natural-Derived Biopolymers. Carbohydr. Polym. 2019, 207 (November 2018), 297e316. [42] Sanchez-Rexach, E.; et al. Sustainable Materials and Chemical Processes for Additive Manufacturing. Chem. Mater. 2020, 32 (17), 7105e7119. [43] Armstrong, C. D.; et al. Enabling Direct Ink Write Edible 3D Printing of Food Purees with Cellulose Nanocrystals. J. Food Eng. 2022, 100451. [44] Jiang, B.; et al. Lignin-Based Direct Ink Printed Structural Scaffolds. Small 2020, 16 (31), 1e10. [45] Sutton, J.; et al. Applications of Polymer, Composite, and Coating Materials Lignin-Containing Photoactive Resins for 3D Printing by Stereolithography Lignin-Containing Photoactive Resins for 3D Printing by Stereolithography. ACS Appl. Mater. Interfaces 2018, 10. [46] Zhang, Z.; et al. Starch Concentration Is an Important Factor for Controlling Its Digestibility During Hot-Extrusion 3D Printing. Food Chem. 2022, 379 (January), 132180. [47] Cosola, A.; et al. Multiacrylated Cyclodextrin : A Bio-Derived Photocurable Macromer for VAT 3D Printing. Macromol. Mater. Eng. 2020, 1e6. [48] Ilhan, E.; et al. Development of Satureja cuneifolia-Loaded Sodium Alginate/Polyethylene Glycol Scaffolds Produced by 3D-Printing Technology as a Diabetic Wound Dressing Material. Int. J. Biol. Macromol. 2020, 161, 1040e1054. [49] Lee, N. A.; et al. Sequential Multimaterial Additive Manufacturing of Functionally Graded Biopolymer Composites. 3D Print. Addit. Manuf. 2020, 7 (5), 205e215. [50] Agarwal, T.; Costantini, M.; Maiti, T. K. Extrusion 3D Printing with Pectin-Based Ink Formulations: Recent Trends in Tissue Engineering and Food Manufacturing. Biomed. Eng. Adv. 2021, 2 (October), 100018. [51] Lapomarda, A.; et al. Pectin-GPTMS-Based Biomaterial: Toward a Sustainable Bioprinting of 3D Scaffolds for Tissue Engineering Application. Biomacromolecules 2020, 21, 319e327. [52] Chen, T. C.; Wong, C. W.; Hsu, S. H. U. I. Three-Dimensional Printing of Chitosan Cryogel as Injectable and Shape Recoverable Scaffolds. Carbohydr. Polym. 2022, 285 (1), 119228. [53] Gu, S.; et al. Chitin Nanocrystals Assisted 3D Printing of Polycitrate Thermoset Bioelastomers. Carbohydr. Polym. 2021, 256 (December 2020), 117549. [54] Saatcioglu, E.; et al. 3D Printing of Osage Orange Extract/Chitosan Scaffolds for Soft Tissue Engineering. Food Hydrocolloids for Health 2021, 1 (November), 100039. [55] Barkane, A.; et al. Thermal Stability of UV-Cured Vegetable Oil Epoxidized Acrylate-Based Polymer System for 3D Printing Application. Polym. Degrad. Stabil. 2020, 181, 109347. [56] Voet, V. S. D.; Guit, J.; Loos, K. Sustainable Photopolymers in 3D Printing: A Review on Biobased, Biodegradable, and Recyclable Alternatives. Macromol. Rapid Commun. 2021, 42 (3), 1e11. [57] Branciforti, D. S.; et al. Visible Light 3D Printing with Epoxidized Vegetable Oils. Addit. Manuf. 2019, 25 (June 2018), 317e324. [58] Miao, S.; et al. 4D Printing Smart Biomedical Scaffolds with Novel Soybean Oil Epoxidized Acrylate; Nature Publishing Group, 2016; pp 1e10. [59] Cui, Y.; et al. 3D Printing of a Dual-Curing Resin with Cationic Curable Vegetable Oil, 2020. [60] Guit, J.; et al. Photopolymer Resins with Biobased Methacrylates Based on Soybean Oil for Stereolithography, 2020. [61] Wu, B.; et al. Direct Conversion of McDonald’s Waste Cooking Oil into a Biodegradable High-Resolution 3DPrinting Resin, 2020.

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CHAPTER 4

3D printing of biopolymer-based hydrogels Fengwei Xie School of Engineering, Newcastle University, Newcastle upon Tyne, United Kingdom

1. Introduction 3D printing creates physical 3D objects based on a 3D digital model by successive addition of materials, contrary to traditionally used subtracted manufacturing. Nonetheless, there is a deficiency of processable, printer-friendly, and environmentally friendly materials (inks) to meet the fabrication and performance requirements, which has constrained the application of 3D printing technologies (1). Given this, the exploration of 3D printable biopolymer hydrogels and the development of 3D printing strategies for them can lead to advanced materials with complex shapes and architecture and appealing functionality. Various 3D printing techniques have been discussed in detail elsewhere (2,3). For hydrogel 3D printing, extrusion-based, inkjet-type, and laser-mediated 3D printing technologies are the most established ones. Nice comparisons in material viscosity, gelation method and speed, preparation time, print speed, resolution, etc. among these different types of 3D printing are provided elsewhere (4,5). Advantages and shortcomings of different 3D printing techniques (6e9) and suitable hydrogels for these techniques (10) are summarized before. This chapter outlines different 3D printing techniques in three categories (extrusionbased, inkjet, and laser-mediated) that are suitable for fabricating biopolymer hydrogels and discusses their material property requirements. In this chapter, some examples of 3D printing of biopolymer hydrogels are highlighted, and future perspectives of biopolymer hydrogel 3D printing are discussed.

2. Biopolymers Natural biopolymers are directly biosynthesized by living organisms (plants, animals, and microorganisms), primarily consisting of polysaccharides (e.g., cellulose, starch, pectin, glucomannan, chitin/chitosan, hyaluronan, alginate, agar, carrageenan, pullulan, gellan gum, xanthan gum (XG), and curdlan) and proteins (soy protein, zein, gluten, casein, collagen, gelatin (hydrolyzed collagen), whey protein, keratin, and silk fibroin) (11). Polysaccharides are carbohydrate polymers formed by connecting monosaccharide units Additive Manufacturing of Biopolymers ISBN 978-0-323-95151-7, https://doi.org/10.1016/B978-0-323-95151-7.00004-1

© 2023 Elsevier Inc. All rights reserved.

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Fig. 4.1 Chemical structures of typical polysaccharides.

via glycosidic linkages. The chemical structures of typical polysaccharides are shown in Fig. 4.1. Proteins are formed by amino acid residues linked via peptide bonds. These biopolymers are readily biodegradable in the natural environment and are especially useful for biomedical applications because all of them are biocompatible and non-toxic and individually they show additional advantages. For example, chitosan is mucoadhesive, antibacterial, and pH-sensitive; gelatin is cell adhesive, biocompatible, and thermoresponsive (3,12). The properties, chemical modification, crosslinking methods, biomedical applications and 3D printing applicability of different biopolymer hydrogels are summarized elsewhere (6,13e15). In the living body, the biodegradability of biopolymers depends on whether there are enzymes that can biodegrade that specific biopolymer. Chemically modified biopolymers may show varied biodegradability as there could be changes to their crystallinity,

3D printing of biopolymer-based hydrogels

chemical structure, hydrophilicity, hydrolyzability, and affinity to specific microorganisms. For example, cellulose acetate especially with a higher degree of substation (DS) may show reduced enzymatic hydrolysis (16). Alginate is naturally non-degradable in mammals, as mammal bodies do not contain the enzyme (i.e., alginase) that can cleave the alginate chain, whereas slightly oxidized alginate can degrade in aqueous media (17). It needs to be emphasized that, by definition, “biopolymers” are usually not equal to “biobased polymers”. The latter group are polymers chemically or biologically synthesized (fully or partially) from biomass monomers, and includes biobased polyesters (e.g., polyhydroxyalkanoates (PHAs), polylatic acid (PLA), polyethylene terephthalate (PET), and polyethylene furanoate (PEF)), polycarbonates, epoxy resins, polyamides, polyurethanes, polybenzoxazines, polyimines, etc. (18). In this sense, only polymers that are biologically synthesized (by microorganisms) based on biomass carbon sources (e.g., sugars and lipids) can be considered as both biopolymers and biobased polymers, and some examples of these are PHAs, bacterial cellulose, gellan gum, XG, and curdlan.

3. Polymer hydrogels Polymer hydrogels are defined in different ways but generally speaking, they are viscoelastic solid-like materials comprised of a 3D network formed by polymer chains crosslinked by covalent bonds, hydrogen bonds, van der Waals force, and/or physical entanglements and water as its main component (or being capable of absorbing and holding considerable amounts of water) (19). Since hydrogels need to be capable of holding water in them, they are formed by hydrophilic polymers, which can be biopolymers as mentioned above (20), and hydrophilic synthetic polymers like polyacrylic acid (PAA), polyacrylamide (PAAm), polyethylene glycol (PEG), and polyvinyl alcohol (PVA) (21). According to their origin, hydrogels can be categorized into synthetic ones, natural ones, and hybrid ones. This chapter focuses mainly on hydrogels based on biopolymers but also involves hydrogels based on combinations of biopolymers and hydrophilic synthetic polymers. According to polymeric composition, hydrogels can be classified into: (1) homopolymeric hydrogels, whose polymeric networks are composed of only one species of monomers; (2) copolymeric hydrogels, which are made of more than one monomer species with at a minimum one hydrophilic constituent, organized in a block, alternating or random configuration along the polymeric chain; and (3) interpenetrating polymeric network (IPN) hydrogels, which are constructed by two separate crosslinked natural and/or synthetic polymeric constituents in a network form. In the third case, there could also be semi-IPN hydrogels, involving a crosslinked polymeric network and a noncrosslinked polymer as the other network (21e24). Fig. 4.2 is a schematic representation of these different types of polymeric hydrogel.

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Fig. 4.2 Schematic representation of (A) homopolymeric hydrogel, (B) copolymeric hydrogel, (C) interpenetrated hydrogel, and (D) semi-interpenetrated hydrogel.

Introducing a secondary polymer network within biopolymer hydrogels renders mechanical enhancement, stimuli-responsiveness, and improved mimicry of the extracellular matrix (ECM). These IPN hydrogels are potential for drug delivery, tissue engineering, and in vitro disease modeling for new drug development (25). Double-network (DN) hydrogels are a distinct type of IPN hydrogel that contains two interpenetrating polymeric networks with contrasting properties. The primary polymer network being highly stiff (i.e., brittle) acts as a sacrificial network, while the secondary, ductile one can be deformed to a great extent. The molar concentrations of the secondary ductile and primary stiff networks are drastically different (20e30 times). The synergistic cooperation of these two polymer networks results in high strength and toughness (25). DN hydrogels have been widely demonstrated to present extraordinary mechanical strength and toughness (26e28). It has been revealed that a local yielding mechanism determines the toughening of DN hydrogels, which shares characteristics with natural nanocomposite materials such as bones and dentin (29). Biopolymeresynthetic polymer DN hydrogels have been widely reported (25). Alginate/PAAm hydrogels (see Fig. 4.3A), as one of the most typical examples of biopolymeresynthetic polymer DN hydrogel, are highly stretchable and exhibit high strength and remarkable toughness (30,33). DN hydrogels based on pure biopolymers have also been reported (25), although to a lesser extent. For example, Sun et al. (31) reported a DN gellan gum/gelatin hydrogel exhibiting impressive mechanical properties and ionic conductivity, which was obtained by treating a gellan gum/gelatin hybrid hydrogel with a Na2SO4/(NH4)2SO4 mixture solution (see Fig. 4.3B). DN hydrogels may offer other enhanced properties than excellent mechanical properties. For example, Karami et al. (32) developed a hydrogel composed of poly(ethylene glycol) dimethacrylate (PEGDMA) and alginate, which could form dual (covalently and ionically, respectively)-crosslinked networks, and nanofibrillated cellulose (NFC) as a reinforcement. This DN hydrogel showed high adhesiveness, advantageous for tissue repair without tissue surface modification (see Fig. 4.3C). Compared to commercial tissue adhesives, this DN hydrogel showed a remarkably increased adhesion strength for soft tissue, e.g., w130 kPa for articular cartilage (32).

3D printing of biopolymer-based hydrogels

Fig. 4.3 Examples of biopolymeresynthetic polymer double-network (DN) hydrogels. (A) The twostep method to synthesize alginate/PAAm hydrogels crosslinked by multivalent cations. (B) The formation of the precursor gellan gum/gelatin gel and DN gellan gum/gelatin gels. (C) The structure of the adhesive hydrogel composed of poly(ethylene glycol) dimethacrylate (PEGDMA), alginate, and nanofibrillated cellulose (NFC). ((A) Reprinted with permission from Ref. (30). Copyright (2013) American Chemical Society. (B) Republished with permission of the Royal Society of Chemistry, from Ref. (31); permission conveyed through Copyright Clearance Center, Inc. (C) Reprinted with permission from Ref. (32). Copyright (2018) American Chemical Society.)

Different types (single-component, IPN, and nanocomposite) of biopolymer-based bioinks for 3D bioprinting have been studied to a great extent, especially in biomedical areas (13,23,34,35). Extrusion-based 3D printing (elaborated in Section 4) is an important method to produce IPN hydrogels, as, after the injection of an ink containing a polymer (e.g., biopolymer) and a polymer precursor, gelation can then be initiated by light, chemicals, or a thermal stimulus to form one more polymer network (26).

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Fig. 4.3, Cont’d

Fig. 4.3, Cont’d

The crosslink junctions in hydrogels can be either chemical or physical. Biopolymers contain abundant functional groups like hydroxyl, carboxyl and amine groups, which can be modified easily for the introduction of crosslinkable groups such as esters, amides, ethers, and carbamates (34). Moreover, different crosslinking strategies can be applied

3D printing of biopolymer-based hydrogels

to biopolymers, such as permanent covalent crosslinking (e.g., click crosslinking, free radical chain polymerization, and crosslinking induced by the oxidation of phenolic groups), dynamic covalent crosslinking (e.g., DielseAlder reaction, Schiff base reaction, and disulfide bond formation), physical crosslinking [e.g., hydrogen bonding, guestehost interactions, metaleligand coordination, and interactions enabled by grafting biopolymer with synthetic polymers such as poloxamers (Pluronic) and poly(N-isopropylacrylamide) (PNIPAAm)] (34). Different types of dynamic bonds such as non-covalent interactions (hydrogen bonding, hydrophobic interaction, ionic interaction, and metaleligand coordination), imine linkage, and DielseAlder reaction-induced linkage can offer polymer hydrogels with not only 3D printability, but also other appealing material properties such as self-healability, high toughness, shape-memory behavior, stimulusresponsiveness, and biodegradability (36). For biopolymers, for example, Alavijeh et al. (37) reported a ureido pyrimidinone hexyl isocyanate (UPy)-modified gelatin hydrogel exhibiting shape memory property useful for controlled drug delivery. Catecholmodified chitosan (38) or hyaluronic acid (HA) (39) hydrogels crosslinked with Fe3þ presented pH-responsiveness. Chemical modification and crosslinking strategies for biopolymer hydrogel 3D printing are nicely summarized in previous reviews (3,13).

4. Extrusion-based 3D printing of biopolymer hydrogels 4.1 Principle of extrusion-based 3D printing The technique of extrusion-based 3D printing involves the use of extrusion as the way to build up pre-defined structures. The extruded material is deposited onto a stage layer by layer, and to create a 3D structure, either the nozzle where the material is extruded out or the stage can be moved (12). The extruded inks can be molten/semi-molten polymers, or solutions, pastes, or dispersions of polymers (8). Compared with inkjet and lasermediated 3D printing technologies, extrusion-based 3D printing shows lower printing resolution and fabrication speed (6); however, extrusion-based bioprinting can handle bioinks with a wide range of viscosity, from 30 mPa$s to 6  107 mPa$s (5). There are two categories of melting-based extrusion printing processes, namely fused deposition modeling (FDM) (or fused filament fabrication (FFF)) (40) and melt electrospinning writing (MEW) (41,42). These approaches are for thermoplastic polymers (e.g., polycaprolactone (PCL), PLA, and PHA), which can soften or melt at an elevated temperature and thus is extrudable but will solidify upon cooling after being deposited on the stage (9,43e45). To ensure the smooth flow of high-viscosity materials, the nozzle for FDM is usually large and thus the extruded fibers are also big, and the printing accuracy is low (42,46). In MEW, the high voltage applied between the spinneret and the collector makes the polymers be extruded charged and deform into a jet whose size is much smaller than the diameter of filaments directly extruded from the nozzle tip (42). In this way, the fiber diameter can be reduced from about 500 to 5 mm (47), which is advantageous for

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biomedical applications. As polymer hydrogels behave differently from thermoplastics and the heat applied may cause the loss of water from the hydrogels, FDM and MEW are generally not suitable for hydrogels. Most hydrogel 3D printing studies are carried out using non-melting extrusion-based printing techniques typically like direct ink writing (DIW). DIW, also known as 3D extrusion, 3D dispensing, or 3D plotting, is capable of printing (extruding) viscous inks such as pastes and concentrated polymer solutions, which subsequently solidify through cooling, drying, chemical reactions, etc. (43). DIW is also an important technique for the 3D printing of foods especially starch-based ones (11,48e53). For successful DIW printing of hydrogels, the printed hydrogels must have suitable and controllable viscoelastic characteristics to enable self-supporting and versatile shaping (further discussed in Section 4.2); Along with this, extruded hydrogels should set instantaneously to allow the printed structures to retain their features. Secondly, a high concentration of nanoparticles or colloids in hydrogels is favored for reducing the shrinkage during the drying of the printed structures (54). DIW can be used for processing diverse ink materials and along with this, the nozzle size used varies from several micrometers to centimeters (43). Generally, the 3D printing of food constructs is achievable with a nozzle diameter of 0.41e2 mm at a printing speed of 2e70 mm/s; for biomedical tissue engineering, 3D printing can be carried out with a 0.15e0.6 mm nozzle diameter at a pace of 0.03e80 mm/s (11). As DIW involves mild fabrication temperature and can easily integrate multiple polymer materials, it is especially useful for bio-fabrication, such as the fabrication of tissue engineering scaffolds and soft robotics (55). For DIW, hydrogels are generally loaded into disposable plastic syringes and then dispensed, either by pressure, or driven by a piston or screw, on a stage (4e6), as shown in Fig. 4.4. The piston-type DIW is more capable of controlling the extrusion flow, while the screw-type one allows greater spatial control and can dispense materials with higher viscosities (4,56). In the literature, “3D plotting” (or freeform 3D printing) may be more specifically referred to as a process of injecting viscous hydrogels into a liquid, paste, or gel (supporting material) with a similar density (54). Generally, the material dispensing head can move in any direction, while the stage is immobile. This method allows the 3D printing of materials with low viscosity that may lack suitable viscoelastic characteristics and selfsupporting ability for a normal DIW process (54). Patrício et al. (57) reported that an XG solution containing Ca2þ is a viscoelastic fluid gel with shear-thinning behavior and thus can act as a continuous pseudo-plastic matrix and a supporting material to enable the rapid fabrication of alginate-based complex 3D structures such as bifurcate tubes (see Fig. 4.5I). In this case, Ca2þ was used for the gelation of alginate once it is printed into the supporting bath (57). Some polysaccharides have inherent gelation behavior upon cooling and heating. For example, both gelatin and agar dissolve in water at an

3D printing of biopolymer-based hydrogels

Fig. 4.4 Three ink-dispensing modes of extrusion-based bioprinting: (A) driven by a piston; (B) driven by a screw; and (C) pneumatically. (Reprinted from Ref. (6).)

elevated temperature and then gelate upon cooling. By taking advantage of this, Landers et al. (59) realized the freeform 3D printing of gelatin- and agar-based scaffolds in a liquid medium with matching density and polarity. Specifically, a 10% concentration gelatin solution at a 40 C cartridge temperature was extruded into silicone oil as the medium at 3 C; and a 5% concentration agar solution was extruded into a 4% concentration gelatin solution as the medium at 20 C (59). Compaan et al. (58) demonstrated that a gellan gum fluid gel can be used as a versatile supporting material for the freeform 3D printing of hydrogel constructs from different materials (see Fig. 4.5II). This gellan gum fluid gel could enable the enzyme-mediated covalent crosslinking of a gelatin/alginate ink, the ionic crosslinking of an alginate ink, physical crosslinking of a gelatin/alginate ink (by temperature drop), and the photo-crosslinking of a poly(ethylene glycol) diacrylate (PEGDA)/gellan gum ink. Samandari et al. (60) designed a DIW 3D printer to realize the continuous printing of alginate/gelatin methacryloyl (GelMA) hydrogel fibers with internal micro-patterns, for controlling cellular orientation in tissue engineering (see Fig. 4.6). Specifically, the printer contained a static mixer, which striated different hydrogels, and a coaxial microfluidic device, where alginate striations were ionically crosslinked by CaCl2 to form fiber. The GelMA striations in the fiber were chemically crosslinked by UV light applied subsequently. The fiber prepared in this way is in millimeters while the internal filaments were on microscale. It was demonstrated that the internal patterned structure realized, which could be tuned by adjusting the flow parameters during printing, could give rise to excellent cell spreading and alignment, rapid cell proliferation and differentiation, and thus the formation of mature tissue engineering constructs.

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Fig. 4.5 (I) Schematic representation of freeform bioprinting using xanthan gum (XG) as a supporting matrix: (A) XG as a sacrificial supporting matrix; (B) XG as a permanent matrix containing a 3D-printed sacrificial internal structure. IOP Publishing. (II) Schematic representation of freeform printing using gellan gum as a supporting matrix: (AeD) show the whole process from filament deposition to post-processing to fabricate a tubular construct. ((I) Reprinted from Ref. (57) with permission from IOP Publishing and the authors. (II) Reprinted with permission from Ref. (58). Copyright (2019) American Chemical Society.)

4.2 Printability evaluation for extrusion-based 3D printing Rheological properties importantly determine the printing resolution and the shape fidelity of 3D-printed constructs (61,62). For extrusion 3D printing, important rheological

3D printing of biopolymer-based hydrogels

Fig. 4.6 Extrusion of alginate/gelatin methacryloyl (GelMA) hydrogel fibers with internal micropatterns for 3D printing: (A) The extrusion setup consisting of a static mixer and a coaxial microfluidic device; (B) simulations of shear stress (s) and pressure (P) in the static mixer and conical nozzles and curves of s/smax and P/Pmax in conical nozzles; (C) fluorescence microscopy images showing the internal microstructure of the extruded fibers. (Reprinted from Ref. (60), with the permission of AIP Publishing, Copyright 2021.)

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parameters include steady-shear viscosity and oscillatory-shear characteristics such as storage and loss moduli (G0 and G00 ), and yield stress (sy) (49,50), which are dependent on the material formulation (e.g., the polymer concentration, chemical structure and molecular mass, and other ingredients) and processing conditions. Especially important to the DIW process, ink formulations must exhibit shear-thinning behavior for easy extrusion out of sub-millimeter diameter nozzles; and once deposited on the stage, the ink must possess high G0 , sy, and low-shear viscosity, for shape retention (to prevent the distortion of printed objects) (63,64). These rheological parameters are explained in the following sub-sections below. 4.2.1 Steady shear rheology As an important rheological parameter, steady shear viscosity (h) describes how easy liquids or gels flow. Polymers, as non-Newtonian fluids, most often show shear-thinning _ as the applied behavior, which means viscosity decreases with increasing shear rate (g), shearing promotes the disentanglement of polymer chains and their orientation along the flow direction (65). To represent this shear-thinning behavior, the linear region of shear rate-viscosity rheology plots can be fitted by a power-law equation (Eq. 4.1) (66): h ¼ K g_ n1

(4.1) 1

where viscosity h is in the unit of Pa$s, shear rate g_ is in the unit of s , n is a dimensionless number called power-law index, and K is consistency (Pa$sn). If n is less than 1, the ink has shear-thinning behavior; n > 1 means the ink is shear-thickening; an n value close to 1 means the ink is similar to a Newtonian fluid (66,67). As mentioned above, shear-thinning is favorable for ink extrusion. Liu et al. (68) found that for a k-carrageenan/XG/potato starch composite gel system, n reduced notably with higher contents of XG and potato starch, particularly at 35 and 45 C, suggesting stronger shear-thinning characteristic. It is noteworthy that the power-law model, as an empirical model, has an applicable shear rate range (typically 101e104 s1), assume the flow is steady without wall slippery, and is only applicable to materials that are incompressible and time-independent (69). To suit practical situations, a wide range of modified forms of the power-law model, as well as other rheological models, have been proposed, which are summarized before (66). After extrusion (shearing), it is crucial for the material to re-gain the initial viscosity before being extruded for achieving ideal shape fidelity. The term, recovery time, means how long it will take for the extruded material to re-gain the initial viscosity, which can be evaluated by altering the shear rate using a rheometer (67,69,70). Much greater printing fidelity can be achieved with materials that naturally recover quickly. Generally, for bioprinting, a recovery time of 5e10 s for gaining >85% of G0 is recommended (67). Kim et al. (71) observed that for alginate/k-carrageenan/CaSO4 hydrogels, an increasing k-carrageenan concentration resulted in higher viscosity with the shear-thinning

3D printing of biopolymer-based hydrogels

Fig. 4.7 Recovery behaviors of different alginate/k-carrageenan/CaSO4 composite hydrogels measured by changing shear rate (stages 1 and 3: 0.1 s1 for 60 s, Stage 2: 100 s1 for 10 s). (Adapted from Ref. (71) with permission from Elsevier, Copyright 2019.)

behavior maintained and the recovery time reduced. For recovery time determination (see Fig. 4.7), the samples were first subjected to shearing in three stages. In the first stage, a rather low shear rate of 0.1 s1 was applied for 60 s, which imitated the stationary state of the sample in a syringe barrel prior to extrusion, and then 100 s1 for 10 s as the second stage to reproduce the state of the materials during extrusion in a nozzle, before going back to the low-shear condition of the first stage; and the time for the viscosity to stabilize in the third stage is determined as the recovery time (71). Viscosity may change with time. Viscosity decreasing with time is termed thixotropy, while rheopectic samples have viscosity increasing with time (67). This viscosity change with time is due to permanent material structural changes caused by shearing. This can be reflected by the recovery test as described above if a material does not recover to its original viscosity following the second high-shear stage (69). Note thixotropy (concerning the effect of a long time of shearing on viscosity) is different from shear-thinning (about the effect of a higher shear rate on viscosity). Materials with strong thixotropy may not be suited for extrusion-based 3D printing as their viscosity is reduced by the extrusion process to a level that negatively affects shape retention. At low shear stress (s), polymer materials behave as elastic solids, but they are prone to flow when the applied s is higher than a critical value named yield stress (sy) (67,72). In steady shear rheology, sy is defined as the critical stress value when the polymer solution/ gel begins to flow, as shown by the first read-out data point with increasing shear rate on a rheometer (73), see Fig. 4.8A. Fluids exhibiting sy are called HerscheleBulkley fluids, whose rheological behavior can be expressed by the following equation: s ¼ sy þ K g_ n

(4.2)

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Fig. 4.8 Different methods to determine yield stress (sy, represented by the black dots). (A) As the intercept obtained by theoretical model fitting, using e.g., the Herschel-Bulkley model. (B) At the cross point of storage and loss moduli (G0 and Gʺ). (C) At a pre-defined deviation (e.g., 85% deviation) of G0 from linearity. (Reprinted from Ref. (67) with permission from Elsevier, Copyright 2019.)

where sy is the yield stress (Pa). With s < sy, the fluid exhibits solid-like behavior and cannot flow; and with the applied s larger than sy, the fluid can then flow, being either shear thinning (n < 1) or shear-thickening (n > 1) (67). sy is considered the most important factor for high printability and shape fidelity. For 3D bioprinting, an sy value of >100 Pa is adequate (67). Mouser et al. (73) concluded that the increased sy of a hydrogel, achieved by the incorporation of gellan gum to GelMA, was the main reason for the improved printability and shape fidelity. With sy known, the minimum pressure (Pmin) in the nozzle required to start the flow of an ink can be calculated by (68,74,75): Pmin ¼

4L sy D

(4.3)

where L represents the nozzle length (m) and D means the nozzle diameter (m). Local shape distortions can be caused by capillary forces as the latter decrease the local curvature of sharp printed edges and overhangs to achieve reduced free energy of the surfaces. If the Laplace capillary pressure (DP) across the surface of a printed layer edge overhang is greater than the sy of the ink, local shape distortion happens. The layer edge will deform to an equilibrium radius (req) and curvature (keq ¼ 1/req) while keq can be calculated from sy and the surface tension (g) of the ink according to the following equation: sy keq ¼ (4.4) g Thus, req ¼

g sy

(4.5)

This correlation speaks of the capillarity-driven mechanism that underlies the local shape distortion of printed filaments, especially when DP is higher than sy. To avoid

3D printing of biopolymer-based hydrogels

too much distortion of the printed material, sy higher than DP is required. And Eq. (4.4) shows how the minimum sy (affected by material formulation and temperature) needed to avoid the shape distortion of printed layer edge overhangs with certain req can be calculated (64). sy can be affected by multiple factors like polymer concentration, the degree of crosslinking, and temperature. For example, Paxton et al. (69) revealed that pre-crosslinked alginate had good printability with a clear yield stress, suitable viscosity, and instant recovery. On the contrary, for the non-crosslinked alginate, there was no clear sy and it took a long time to recover. For materials with thermo-reversible gelation behavior (e.g., k-carrageenan), temperature also influences sy. Liu et al. (68) revealed that the sy of an ink formulated with 1 wt% k-carrageenan, 0.5 wt% XG, and 2 wt% potato starch was 553.1 Pa at 35 C and declined to 36.9 Pa 45 C, showing its temperature-dependence. For a k-carrageenan (1 wt%)/XG (0.5 wt%) sample (containing no starch), a higher temperature caused the ink to behave like liquid and be unable to form self-supporting layers (68). Liu et al. (50) showed the sy (from 44.41 to 883.19 Pa) of a potato starch paste increased with a higher starch concentration (from 10 to 30 wt%), and higher sy was favorable for higher printing accuracy and higher strength of the printed structure. Moreover, to increase the sy for hydrogels, an easy route is the incorporation of micro-/nanoparticles, which decreases the available space within the hydrogel leading to a thickening effect (67). 4.2.2 Oscillatory shear rheology In dynamic rheology, storage modulus (G0 ) represents the elastic solid-like behavior of a material while loss modulus (G00 ) is used to describe materials’ viscous response (65). It is beneficial for the hydrogel ink to have liquid-like characteristics (G00 > G0 ) during printing while gel-like characteristics (G0 > G00 ) after printing. In addition, whether a material is liquid-like or gel-like can also be reflected by loss tangent (tan d ¼ G00 /G0 ) (76). Specifically, tan d > 1 (phase angle 45 degrees < d < 90 degrees) generally means that the ink has the viscous nature more dominant and is flowable and extrudable, whereas tan d < 1 (0 degrees < d < 45 degrees) indicates the ink possesses an elasticity-dominant gel structure (77,78). Gholamipour-Shirazi et al. (76) evaluated the extrusion 3D printability of different food hydrocolloid formulations with different base materials (including polysaccharides and gelatin) and concentrations, and their results indicate a correlation between phase angle and printability. Specifically, liquid and non-self-supporting inks exhibited high d values (45e90 degrees at 1 Hz); semisolid but non-self-supporting inks showed medium d values (15e45 degrees at 1 Hz); medium-low d values (3e15 degrees at 1 Hz) was associated with semisolid and self-supporting inks; and when the d was low (0e3 degrees at 1 Hz), the material was hard to extrude (76). The polymer concentration in the ink and printing temperature determines how easy molecules move and the extent of chain entanglement and interactions and thus the viscoelasticity of extruded materials. For example, Roehm and Madihally (79), who

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studied the rheological properties of chitosan/gelatin hydrogels as affected by temperature and concentration for 3D printing purposes, found that increases in the concentrations of chitosan and gelatin led to higher G00 , particularly at lower temperatures. Besides, a higher temperature (from 26 to 47  C) increased both G0 and G00 increased notably (79). Besides, the viscoelasticity of inks may also be modified by inclusion of micro-/ nanofillers. For instance, Markstedt et al. (80) investigated the effect of the NFC/alginate ratio (9:1, 8:2, 7:3, and 6:4, w/w) on the oscillatory rheological properties of bioinks. Increasing NFC proportion in the bioink was found to cause higher G0 and G00 . Furthermore, all the bioinks had tan d lower than 1, suggesting their solid-like behavior (80). As mentioned in Section 4.2.1, sy can be determined by fitting with empirical models (e.g., Herschel-Bulkley). Besides, the apparent sy of an ink can be taken as the cross point of the G0 and G00 curves in dynamic rheology (64,67,68) (see Fig. 4.8B). Moreover, sy can be determined by the shear stress at a pre-determined deviation of G0 from linearity (67) (see Fig. 4.8C). 4.2.3 Other aspects The consistency of bioinks during extrusion printing needs to be evaluated by monitoring the force fluctuation. For example, Chung et al. (81) found that, compared with water and the 4% alginate/gelatin ink (which were similar to each other), the 4% alginate/Ca2þ ink led to greater fluctuation in extrusion force, suggesting a higher degree of heterogeneity of the ink. 4.3 Solidifying process for extrusion-based 3D printing Solidification allows the deposited layers to have adequate strength to hold the weight of their own and following layers (82). Solidification can be realized by the gelation or crosslinking of biopolymers, before and/or after printing. The solidification of hydrogel-forming polymers can be achieved using physical crosslinking, enzymatic crosslinking, and/or chemical crosslinking, which are exemplified in the following subsections. Many crosslinking methods have been explored to create (bio)polymer hydrogels (34,35), which have not been all used to solidify 3D-printed biopolymer hydrogels but could inspire future work in this area. 4.3.1 Physical crosslinking Some biopolymers exhibit gelation behavior that can be induced by physical (e.g., temperature and pressure) or chemical (e.g., pH, ionic, and enzymatic) conditions, depending on different mechanisms (non-covalent interactions such as hydrophobic and ionic interactions, hydrogen bonding, coilehelical transition, chain aggregation, and metale ligand coordination, and covalent bonding such as disulfide bond formation in some protein gels) (83).

3D printing of biopolymer-based hydrogels

The temperature-dependent gelation behavior of biopolymers can be either thermoreversible or thermo-irreversible. Thermo-reversible biopolymers include cold-set gels (e.g., agar (containing agarose), carrageenan, gellan gum, and gelatin) and heat-set gels (e.g., methylcellulose, hydroxypropyl methylcellulose (HPMC), low-set gel of curdlan (set at a relatively low temperature), xyloglucan); thermo-irreversible gels are those, once formed on heating, will not melt, such as konjac glucomannan (with the existence of alkali which has a deacetylation function), high-set gel of curdlan (set at a relatively high temperature), whey protein, and egg protein (83e85). For cold-set biopolymer gels, deposition of such materials hot onto a cool stage allows them to cross their gel transition temperatures and solidify. For example, a high temperature (60e80 C) of the printer reservoir was necessary to maintain the liquid form and extrudability of an agarose-solution ink, while a cool bath (below its gel transition temperature) where the ink could be printed into was employed to solidify the ink (59,86). In another study, a bioink based on two species of silk fibroin (Bombyx mori and Samia cynthia) with self-gelling ability and gelatin for bulking purposes was developed, which had satisfactory shape fidelity for cartilage (e.g., human ear) tissue engineering (87) (see Fig. 4.9I). The strength of hydrogels formed by simple gelation is usually weak and needs to be further improved by supplementary solidification strategies such as ionic or chemical crosslinking (12). Ionic crosslinking occurs by means of electrostatic interaction between two reversely charged molecules (cations and anions); and for gel formation, at least one of the molecules has to be a polymer (polycation or polyanion) (12). For example, alginate, a polyanion comprising mannuronic and guluronic acid residues, can be crosslinked ionically to form a hydrogel with the existence of divalent cations such as Ca2þ. It is believed divalent cations can only interact with guluronate blocks of the alginate chain (17). This feature of alginate has been widely employed to construct alginate-based hydrogels (89). The gelation of printed alginate can be induced by Ca2þ in a supporting medium (58) (see Fig. 4.9II)) or after printing (for IPN hydrogels) (90). Besides, pre- (partially) crosslinked alginate showed sufficient stability to maintain its shapes after printing (69). Chitosan is another example that can undergo ionic crosslinking. Above its isoelectric point, chitosan is positively charged (due to the transformation of NH2 groups on the glucosamine residues into NHþ 3 groups) and can be crosslinked with phosphate ions when being heated (91). Moreover, ionic crosslinking of low-methoxyl pectin occurs with an appropriate concentration of CaCl2 (92,93). Phenolic compounds including phenolic acids have the capability of crosslinking polysaccharides and proteins (94). Tannic acid, a polyphenol extracted from plants, has received a strong focus recently for the one-pot preparation of hydrogels since it can form extensive hydrogen bonding with biopolymers like gelatin (95,96) and carboxylated agarose (97). For the 3D printing of porous collagen scaffolds laden with cells, different concentrations of tannic acid (0.1e3 wt%) were tested for hydrogen-bonding

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Fig. 4.9 (I) Schematic illustration of (A) the formulation of the silk fibroin/gelatin bioink, (B) the entanglement and interaction between silk fibroin and gelatin within the bioink, and (C) the digital modeling and (D) the 3D bioprinting of a cell-laden construct. (II) Calcium-mediated ionic crosslinking of an alginate-based ink (solution of 2% sodium alginate in phosphate-buffered saline): (A) alginate crosslinking by Ca2þ and (B) resulting alginate structures (scale bars: 5 mm). (III) Schematic representation of the fabrication of MC3T3-E1-laden collagen scaffold without and with crosslinking using tannic acid (concentration 0.1e3 wt%). ((I) Reprinted with permission from Ref. (87). Copyright (2019) American Chemical Society. (II) Reprinted with permission from Ref. (58). Copyright (2019) American Chemical Society. (III) Reprinted from Ref. (88) with permission from Elsevier, Copyright 2018.)

crosslinking for 10 min; and the sample crosslinked with 0.5 wt% tannic acid showed largely increased mechanical strength and biocompatibility (88) (see Fig. 4.9III). 4.3.2 Enzymatic crosslinking Enzymes such as transglutaminases, tyrosinases, laccases, and peroxidases are usually used for crosslinking protein-based materials (94,98). Enzymatic crosslinking is profoundly useful for creating hydrogels for tissue engineering considering its moderate reaction conditions, high efficiency (7e20 min), and notable biocompatibility (99,100). It has been demonstrated that enzymatic crosslinking can improve the rheological characteristics and printability of bioinks (101,102). For instance, Chameettachal et al. (101) manifested that tyrosinase can be used as a crosslinking agent to enable the 3D printing of cartilage

3D printing of biopolymer-based hydrogels

constructs based on a silk fibroin/gelatin bioink. The constructs prepared in this way could improve the survivability of cells in vitro and lessen hypertrophy (101). According to Zhou et al. (102,103), Ca2þ-independent microbial transglutaminase could be used to catalyze the formation of isopeptide bonds between GelMA chains to increase the gel viscosity. To avoid excessive crosslinking that would negatively impact the printability of the hydrogel, the enzyme was deactivated subsequently. Post-printing photocrosslinking was applied to endow the printed construct with enduring stability for the following cell studies (102,103). Similarly, Petta et al. reported a tyramine-modified hyaluronic acid-based bioink established by dual crosslinking, where an enzymatic reaction realized by horseradish peroxidase (HRP) along with hydrogen peroxide (H2O2) resulted in a soft gel appropriate for the encapsulation of cells and for extrusion, while the shape retention of the printed structures was achieved by visible-light photo-crosslinking. Markstedt et al. (104) prepared stable gels by mixing tyramine-functionalized xylan with NFC and crosslinking with HPR and H2O2. The printability and crosslinking density of the gel ink were found to be affected by the level of tyramine substitution and the ratio of NFC to the tyramine-functionalized xylan. The incorporation of the crosslinkable xylan in NFC dispersions led to all-wood-based inks that, after being 3D-printed, can form free-standing gels (multi-layered gridded structures), while the excellent printing properties of NFC were not jeopardized. 4.3.3 Chemical crosslinking Photoinitiation is a widely used approach to generating free radicals for hydrogel formation (105). A polymer under irradiation undergoes photolysis (namely the breakage of some bonds and the generation of free radicals) and between activated molecules, subsequent reactions occur and thus new bonds form leading to a network structure, which is referred to as photo-crosslinking (106). For free-radical polymerization, biopolymers can be functionalized with methacrylate groups. This can be achieved in various ways such as the esterification of biopolymers with methacrylic anhydride and etherifying biopolymers with glycidyl methacrylate (GMA) (34). The most commonly seen modified biopolymer for 3D printing is GelMA (73,102,103,107e118). For extrusion 3D printing, other biopolymers have also been functionalized with methacrylate moieties to be UVcurable, such as alginate (60,119), chitosan (118,120), and HA (121). For example, Ouyang et al. (121) reported a 3D-printable hyaluronic acid-based hydrogel ink with both shear-thinning behavior and self-healing ability. A double crosslinking strategy was used to create this ink, involving guestehost bonding (achieved by modifying HA with adamantane and cyclodextrin) and covalent photo-crosslinking for stabilization (achieved by modification of HA with methacrylic anhydride) (see Fig. 4.10A). According to Cao et al. (119), a 3D-printed methacrylated alginate (SA-MA) hydrogel was cured during printing under UV light, and the succeeding ion exchange with Ca2þ ions and ionic interaction with chitosan could stepwise raise the crosslinking density, increase

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Fig. 4.10 (A) Different hyaluronic acid (HA) derivatives that are crosslinkable and useful as inks for 3D printing, including methacrylated hyaluronic acid (MeHA) which is UV-curable, adamantane-coupled hyaluronic acid (Ad-HA)/b-cyclodextrin-coupled hyaluronic acid (CD-HA) for guestehost (GH) assembly, and adamantane-coupled methacrylated hyaluronic acid (Ad-MeHA)/b-cyclodextrin-coupled methacrylated hyaluronic acid (CD-MeHA) for dual-crosslinking (DC). (B) (a) Reaction scheme for methacrylated alginate (SA-MA); (b) Schematic illustration of the formation of multi-crosslinked methacrylated alginate. ((A) Reprinted with permission from Ref. (121). Copyright (2016) American Chemical Society. (B) Reprinted with permission from Ref. (119). Copyright (2021) American Chemical Society.)

3D printing of biopolymer-based hydrogels

the mechanical properties, and cause a volume contraction of the hydrogel (see Fig. 4.10B). Since, during printing, different parts of the material were irradiated to different extents, the 3D-printed hydrogel had a gradient pattern of crosslinking, which allowed it to exhibit multiple deformations via two-step ionic immersion. In addition, a bilayer of SA-MA hydrogel with varied densities of chemical crosslinking was prepared, which, upon Ca2þ soaking, showed an anisotropic shape change. Heidenreich et al. (122) demonstrated that for the 3D printing of collagen/chitosan blend gels, N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) added in the precursor stage was a viable route to mechanically strengthening the final construct. As NHS/EDC allows covalent bonds to form between carboxyl ( COOH) or phosphate groups (OPO(OH)2) and primary amines (NH2), covalent amide links could be established both between collagen chains and between chains of collagen and chitosan. For IPN biopolymer/synthetic polymer hydrogels, chemical crosslinking to form a synthetic polymer network is important to achieve excellent mechanical properties. Bakarich et al. (90) reported the large-scale production of tough gels with complex shapes using extrusion printing accompanied by photo-polymerization. The printed alginate/ AAm ink was treated with UV light for the gelation of AAm to form PAAm, leading to good mechanical properties (tensile strength of 100e200 kPa, breaking elongation of 300%e400%, and Young’s modulus of 40e80 kPa). Immersing and swelling the printed gels in a CaCl2 solution further increased its elastic modulus to w370 kPa, while their original shapes could still be retained, because of the formation of ionic crosslinks. In contrast, the printed gel swollen in water had its geometrical shapes lost and extremely poor mechanical properties (90). Similarly, Liu et al. (123) reported the 3D printing of a k-carrageenan/AAm solution ink accompanied by UV-induced polymerization to create complex-shaped structures (a hollow triangular prism and a hollow cube) with notable mechanical strength. This hydrogel has a k-carrageenan network ionically crosslinked with KCl combined with a covalent PAAm network crosslinked using N,N0 methylenebisacrylamide (MBA). This k-carrageenan/PAAm DN hydrogel was highly sensitive to strain, with a gauge factor of 0.63 at a 1000% strain, and therefore, can be applied as strain-sensors for applications of robotics and human-motion detection (123).

5. Inkjet 3D printing of biopolymer hydrogels 5.1 Principle of inkjet 3D printing Inkjet 3D printing represents a category of techniques to eject and deposit solventdispersed materials on a substrate via a nozzle (3). Only liquids with low viscosities (3.5e 12 mPa$s) can be inkjet-printed (5). Inkjet 3D printing can be operated in either the continuous or drop-on-demand (DOD) mode, with the latter one more widely used (124). The dispersed material after being printed out is then solidified by UV light or

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Fig. 4.11 Thermal and piezoelectric inkjet bioprinting. (Reprinted from Ref. (6).)

chemical or ionic crosslinking (125). In the continuous mode, the fluid is driven through the nozzle continually and the jet subsequently breaks into a flow of droplets resulting from capillary-driven Rayleigh-Plateau instability. In the DOD mode, drops are only produced as required by means of thermal or piezoelectric actuation (15,124) (see Fig. 4.11). In thermal DOD 3D printing, heat is applied to produce vapor bubbles, which eject ink droplets forcefully. In piezoelectric DOD 3D printing, there is a piezoelectric element that can deform to cause acoustic pulses, which push the ink droplets out of the nozzle (126). For thermal DOD 3D printing, inks must be relatively volatile, or at least have a volatile component, while there is no such restriction for the piezoelectric DOD method (127). Inkjet 3D printing has proven to be a highly valuable tool in life sciences and is especially useful for drug discovery considering its reproducibility and full automation (3). Thermal inkjet printing and piezoelectric one are the two most frequently employed approaches for the inkjet printing of cells (4,5). 5.2 Biopolymer hydrogel materials fabricated by inkjet 3D printing There have been limited studies on the inkjet 3D printing of biopolymer materials. Xu et al. (128) reported the rapid production (55,000 particles/s) of single-cell microparticles (30e60 mm) by inkjet printing. Specifically, using a normal thermal inkjet printer, alginate microparticles containing one to several insulin-producing cells (b-TC6) were fabricated by co-printing the cells and sodium alginate suspension into a CaCl2 solution. Ferris et al. (129) devised a microgel bioink formed on gellan gum and Dulbecco’s Modified Eagle Medium (DMEM), added with surfactants (Poloxamer 188 surfactant and/or fluorosurfactant), for DOD bioprinting (droplets dispensed by a magnetic feedback-

3D printing of biopolymer-based hydrogels

controlled microvalve or a piezoelectric inkjet print head) of different types of cells. The bioink maintained a stable suspension of cells free of settling and aggregation, which is vital for cell printing, meanwhile satisfying the rigorous requirements of fluid properties and printability even for commercial inkjet print heads with many nozzles. However, in both of these studies, no 3D constructs were realized (129). Xu et al. (130) reported the 3D inkjet printing of complex 3D constructs like zigzag tubes using a bioink composed of sodium alginate (2%, w/v) mixed with a cell suspension at a 1:1 (v/v) ratio. The print head, which could move horizontally, jetted the bioink into a bath containing a CaCl2 solution for ionically crosslinking the alginate while the printed structure was formed on a platform that is vertically movable (see Fig. 4.12A). Fibroblast (3T3 cell)-laden tubular and overhang-structured constructs with high post-printing 3T3 cell viability (above 82% after an incubation period of 72 h) were obtained (130). In another study, Xu et al. (131) fabricated complex-shaped and heterogeneous tissue constructs using a modified thermal inkjet printer to jet multiple types of cells concurrently (see Fig. 4.12B). In the printer, there are separate ink cartridges loaded with 0.1 M CaCl2, solutions mixed with human amniotic fluid-derived stem cells (hAFSCs), canine smooth muscle cells (dSMCs), and bovine aortic endothelial cells (bECs), respectively. A mixture solution of sodium alginate and collagen was used as a supporting material for the printing and CaCl2 acted as a crosslinker for sodium alginate to form hydrogel. In-vitro and invivo evaluations indicated that the cells in the printed construct had high viability and could proliferate effectively and grow into tissues with sufficient vascularization (131). These findings show that it is the potential of biopolymers and inkjet 3D printing to create complex tissues constructs.

Fig. 4.12 Schematic representations of two 3D-inkjet-bioprinting systems: (A) platform-assisted, with a sodium alginate-based bioink; (B) with a sodium alginate/collagen mixture-based bioink to inkjet multiple cell types simultaneously. ((A) Reprinted from Ref. (130) with permission from John Wiley and Sons, Copyright 2012. (B) Reprinted from Ref. (131) with permission from Elsevier, Copyright 2013.)

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6. Laser-mediated 3D printing 6.1 Principle of laser-mediated 3D printing Laser-mediated 3D printing techniques rely on light to induce polymerization or crosslinking of polymers to fabricate 3D structures. Given this, different types of light typically as UV, infrared, and even visible light can be used (15). Laser-mediated 3D printing techniques for hydrogel fabrication include stereolithography (SLA) and two-/multi-photon polymerization, which are categorized as vat photopolymerization techniques (3). Compared with other 3D printing techniques, SLA is more capable of controlling the dimension and structural details of the printed 3D constructs with higher resolution (132). There is a limitation to the viscosity of inks suitable for SLA, 1e300 mPa$s (5). Compared with extrusion and inkjet bioprinting, SLA-based one can be used to fabricate complex-shaped tissue structures with higher resolution and overall quality but lower cost (6). SLA 3D printing generally is set up with a vat (reservoir), a movable platform (stage), a photocurable resin (liquid), and a light source (UV generator or LED) as shown in Fig. 4.13A (133). In the bath configuration (see Fig. 4.13A(a)), the stage is positioned just beneath the top surface of the resin in the vat and the layer of resin above the stage is the targeted one to cure first. The curing of this layer of resin is performed by a sole laser that irradiates it point by point (point-type curing) and moves row by row, until the whole area is fully cured. For curing the succeeding layer, the position of the stage is lowered to allow a suitable amount of the resin to cover the top surface of the previous layer, and then the curing process repeats (133). In a layer configuration (see Fig. 4.13A(b)), the stage is submerged in the photopolymer reservoir for a defined distance. Next, a laser

Fig. 4.13 (A) Schematic representation of two typical configurations of stereolithography (SLA) 3D printing: (a) bath configuration with point-type curing; (b) layer configuration with projection-type curing. (B) Schematic representation of two-photon photopolymerization (2PP) 3D printing. ((A) Adapted with permission from Ref. (133). Copyright (2014) American Chemical Society. (B) Reprinted from Ref. (10) with permission from Elsevier, Copyright 2012.)

3D printing of biopolymer-based hydrogels

from the bottom is guided to the stage to cure the resin under the stage in the vat. In the projection-type curing method, the digital mirror device underneath the vat can guide the laser to irradiate the whole area of the layer to cure it at the same time. Afterward, the stage is raised by a defined distance to allow another layer beneath the previously cured layer to be cured (133). This second configuration is also called digital light processing (DLP). As in DLP, a masked light source is employed to cure a whole layer straight away but not point by point, the printing efficiency is appreciably higher than using the bath-configuration SLA (8,9). Thereby, DLP is less influenced by oxygen inhibition since the layer of resin being cured is always in the vat without being in contact with the air) (9). DLP can provide a lateral resolution between 10 and 50 mm (8). Two photon-polymerization (2PP) is a recently developed laser-based curing method in 3D printing. While other light-curing methods are based on a single photon, the 2PP one realizes the photo-induce polymerization or crosslinking by using near-infrared femtosecond laser pulses, which, being excited in a nonlinear way, cure the resin only at the focal point without affecting other regions (Fig. 4.13B). Moving the laser focus allows the curing of the resin in different regions and in this way, a 3D object can be fabricated (10). The term “multiphoton” describes three or more photons that are simultaneously absorbed by the resin (despite being at very low probabilities) for photoinduced polymerization or crosslinking (8). The 2PP technique can be used for the high-resolution fabrication of complex-shaped 3D structures at micro- and nanoscales (134e136). This technique is capable of constructing micron-sized objects containing features in the size of 99% can be achieved within 40 s (151) (Fig. 12.20) 5.4 Soft robotics/smart actuators Another interesting application is for the design and fabrication of soft robotics and smart actuators. A wide variety of advanced materials with various functionalities have been

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Fig. 12.18 (A) DMA curve of c-PCL; (B) shape fixity and recovery ratios of c-PCL; (C) DSC heating and cooling profile of c-PCL; (D) schematic diagram of scaffold shape memory process. (Adapted with permission (149).)

developed to fabricate highly complex designs using 4D printing that enables the smart printed structures to evolve over time in response to environmental stimuli. A novel bilayer is printed using SLA printer to fabricate two distinct layers of poly(N,N-dimethyl acrylamide-co-stearyl acrylate) (P(DMAA m-co-SA))-based hydrogels containing different concentrations of the crystalline hydrophobic monomer (SA) within the shape memory hydrogel (SMG) network (Fig. 12.21). It exhibited anisotropic swellinginduced actuation of the bilayer with shape memory properties when exposed to water and has the ability to unfold and return to its original shape upon heating (157) (Fig. 12.22). Another study utilized extrusion-based printing to fabricate 4D printed grippers using composite polylactic acid (PLA)/silver nanowires (Ag-NWs) (158). The presence of the Ag-NWs network conferred the excellent electric conductivity to induce localized deformation within the printed actuators upon exposure to electrical stimulation after repeated tensile and bending deformations. Furthermore, it has demonstrated that the applied voltage can be used to control the deformation of each petal of a printed biomimetic flower independently.

Fig. 12.19 Fabrication process for the shape memory airway stent. (A) Digital model of a tracheobronchial tree from MRI scan, (B) a CAD model of the structure, (C) printing process using stereolithography (SLA) for the fabrication of shape memory airway stent, (D) printed tracheal stents in the permanent shape, (EeF) images showing the transition between the temporary state (for deployment) and the permanent shape. The time taken for the stent to completely open was 14 s. (Adapted with permission (155).)

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Fig. 12.20 Representative images of the shape recovery process for the 4D printed composite tracheal stent. (Adapted with permission (151).)

Fig. 12.21 (A) Bending deformation mechanism of the bilayer composed of 3D printed SMG70-SA30 and SMG90-SA10, (B) at room temperature (50 mm length, 1 mm thickness), (C) at 60 C (50 mm length, 1 mm thickness), (D) at 60 C (50 mm length, 2 mm thickness), (E) at 60 C (20 mm length, 1 mm thickness), (F) temperature-dependent curvature during recovery (50 mm length, 1 mm thickness) as a function of time, and (G) recovery process of the bilayer in water at (i) 50 C, (ii) 60 C and (iii) 70 C, indicating faster recovery with increased temperature. (Adapted with permission (157).)

Potential applications of 3D and 4D printing of biopolymers

Fig. 12.22 4D printed composite PLA/ag-NWs smart gripper (A) a biomimetic flower with five petals, and each petal can be independently expanded under the influence of applied voltage, (B) a gripper grabbed a ball from icy cold environment. (Adapted with permission (158).)

6. Conclusion Biopolymers made from proteins, polysaccharides, and synthetic biopolymers play a critical role in tissue engineering and regenerative medicine, drug delivery, and biomedical applications. In this book chapter, the three main classifications of 3D printing techniques (including material extrusion, material jetting and vat polymerization) for biopolymers are investigated. Having a good understanding of the printing process and material requirements is important for printing complex 3D structures with good printability and accuracy. Although 4D printing is still in the infancy stage, proof-of-concept studies have demonstrated the potential of 4D printing for various applications such as vascularization in tissue-engineered constructs, controlled drug delivery, and smart biomedical devices such as 4D printed stents. However, there are existing limitations to 4D printing that needs to be addressed. The current state-of-the-arts 4D printing is currently suitable for small, foldable objects. The first key limitation is the limited choice of biopolymers

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suitable for 4D printing applications as most biopolymers are not responsive to exogeneous stimulation. Next, most of the mechanisms for 4D printed structures require physical contact (temperature, moisture, pH, ions etc) and contactless stimulation for 4D printed structures is an emerging research field that will be advantageous for non-invasive biomedical applications. Lastly, most of the 4D printed structures can only be used to achieve relatively simple programmed structures, more research could be done in the near future to enable precise control and tunability of 4D printed structures to achieve more complex 3D structures. To sum up, more in-depth investigation is required to precisely control the programmed change in shape, property, or functionality of stimuliresponsive material on a microscale level.

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CHAPTER 13

3D printing with biopolymers: toward a circular economy Alysia Garmulewicza, b, f, Filippos Tourlomousisc, d, e, Charlene Smithf and Pilar Bolumburuf a

Department of Management, Faculty of Management and Economics, University of Santiago of Chile, Santiago, Chile; CABDyN Complexity Centre, University of Oxford, Oxford, United Kingdom; cNational Centre for Scientific Research Demokritos, Attica, Greece; dSuperlabs AMKE, Marousi, Greece; eBiological Lattice Industries Corp, Boston, MA, United States; fMateriom, London, United Kingdom b

1. Introduction The transition to a circular economy is a growing imperative. Over the past decade, the concept has gained traction among business, policy, and academia as a vision for an economic and industrial system that could overcome many of the resource challenges experienced today (1). The model is commonly portrayed as comprising a set of strategies to maintain and circulate products, components, and materials in the economy, within biological and technical cycles (2). The biological cycle comprises strategies for materials that contain valuable nutrients for the biosphere and thus can be cycled via natural systems through composting and biodegradation. The technical cycle aims to maintain materials valuable to the industry in a closed-loop, where products, components, and materials are shared, repaired, reused, remanufactured, and eventually recycled within the industry. Here, we explore the relationship between 3D printing and the circular economy through the lens of 3D printing biopolymers. 3D printing is defined as “a process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies” (3). 3D printing is often used synonymously with additive manufacturing (AM). 3D printing biopolymers can be considered as a specific lever of circular economy innovation. This can be understood as part of the research agenda on material supply chains for 3D printing and the circular economy. Elements discussed here, biopolymer sourcing and life-cycle analysis, have implications for 3D printing as it relates to circular economy principles, including product design, information flows, and entrepreneurial responses. The chapter is organized as follows. Continuing part one, the circular economy is introduced as an organizing concept for product, process, and production system innovation. We then introduce the role biopolymers play in a circular economy. In part two, we provide a technical introduction to 3D printing with biopolymers. In parts three to five, we analyze the potential for 3D printing biopolymers to enable the circular economy at three levels: material, process, and supply chain, providing a nested view of Additive Manufacturing of Biopolymers ISBN 978-0-323-95151-7, https://doi.org/10.1016/B978-0-323-95151-7.00008-9

© 2023 Elsevier Inc. All rights reserved.

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innovation. At the material level, performance and functionality are being added by adding 3D structure to biological building blocks. This opens the possibility of greater market application and future substitution of today’s polluting industrial materials. At the process level, we review life cycle assessments to better understand the potential process improvements that can be made. At the supply chain level, we discuss how to source biopolymers in a way that does not degrade ecological systems, instead helping to create a regenerative circular economy. We then provide a case study on sourcing chitin from crustacean waste, providing a window into possibilities for sourcing 3D printing biopolymers from locally abundant biomass. A discussion of research horizons concludes the chapter. 1.1 Introduction to the circular economy Since the Industrial Revolution, the global economy has been dominated by a linear model of production and consumption, in which goods are manufactured from raw materials, and sold to consumers, who then use and discard them as waste (4,5). This linear production-consumption model has resulted in exponential growth of negative social and environmental externalities, including dangerous levels of greenhouse gas emissions, land-use change, and novel entities from chemicals and plastics (6,7). With the global middle class projected to grow from 3.2 billion in 2015 to 5.4 billion by 2030 (8), these trends are likely to continue. Considering the impact of chemicals and plastics, the production of chemicals is projected to triple by 2050 (7), with plastics set to account for 20% of oil production (9). This underscores the critical need to explore material alternatives such as biopolymers for manufacturing across product niches including packaging, textiles, and household goods. The concept of circular economy has emerged from various strands of thought and research as a potential vision for an economic and industrial system that can regenerate and work within the earth systems that support humanity. The roots of circular economy as a concept are attributed to Boulding (10) for his work on the “Economics of the Coming Spaceship Earth”, Stahel and Reday (11) for their conception of industrial economics, Stahel (12) for introducing the idea of the performance economy, Benyus (13) for the biomimicry perspective and McDonough and Braungart (14) for the Cradle-to-Cradle framework. Papers providing a review of the historical roots of circular economy in the scientific literature include Anderson (15), Su et al. (16), Ghisellini, Cialani, and Ulgiati (1), and Geissdoerfer (17). 1.2 3D printing and the circular economy 3D printing has been identified as a potential enabling technology for the circular economy (18). However, there are numerous outstanding questions as to how emerging value chain configurations, stakeholder relationships, and product and material lifecycles of 3D printing related to circular economy goals and principles (19). Further research is needed on how 3D printing and circular economy align regarding product, service, and system

3D printing with biopolymers: toward a circular economy

design, material supply chains, information structure and flows, entrepreneurial responses, business model transformations, and education and skills development. For example, on the design dimension, a key question is how 3D printing processes and resulting products can enable circular economy principles such as re-use, modularity, upgrade, refurbishment, and remanufacture (19). Unruh (18) has identified a number of characteristics of 3D printing that align with principles that enable a healthy and functioning biosphere. The principles identified are using a parsimonious materials palette, creating power autonomy through renewable energy use, value cycling of materials, sustainable product platforms that can leverage economies of scale and scope, and building form to function. 3D printing was found to enable many of these, such as using a single material to build multiple forms and functions, having 3D designs that can proliferate on product platforms, and enabling the precise manipulation of form to achieve the desired function. The primary misalignment of 3D printing technology with biosphere principles and circular economy is materials parsimony, in terms of the number of molecular building blocks used, and the value cycling of these materials. Aside from metal 3D printing, the majority of materials used are petrochemical plastics such as Nylon, and acrylonitrile butadiene styrene (ABS). There have been some efforts to develop closed-loop systems with recycled plastic for 3D printing (20,21), with studies showing the potential disruptive impact at a systems-level of manufacturing with local plastic waste streams (20,22). Companies such as Reflow have successfully commercialized filaments from recycled Polyethylene terephthalate glycol (PETG), polylactic acid (PLA), Poly (methyl methacrylate) (PMMA), and polypropylene (PP) (23). Despite these efforts, the majority of 3D printing plastics are not designed to be part of a circular economy. Here we examine how 3D printing biopolymers, which are made from a select set of elements - carbon, hydrogen, oxygen, and nitrogen - can enable a circular economy. 1.3 Biopolymers as part of the biological cycle Materials in the technical cycle are commonly sourced from finite resources, such as metals, minerals, and petrochemical reserves. Materials in the biological cycle, by contrast, are typically renewable and globally abundant. Materials are part of the biological cycle if they can re-enter the biosphere safely while providing nutrient value to living organisms. Most materials in the biological cycle are biopolymers: defined as being sourced directly from renewable biomass synthesized by natural organisms, including plants, animals, fungi, and bacteria. Natural organisms compose biomaterials from various carbohydrate, protein, fat, and phenolic molecular building blocks, as well as select minerals like calcium carbonate and biosilica (24e26). Biopolymers such as cellulose, hemicellulose, lignin, and chitin, are some of the most abundant materials in the biosphere. Cellulose, constituting around 50% of plant matter, is estimated to be produced at two teratons (212 tons) in the biosphere annually, making it

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the most abundant renewable material on Earth (27). Chitin, hemicellulose, and lignin are estimated at 100 billion, 60 billion, and 10 billion tons respectively in annual production volume (28). To put these volumes in perspective, 8.3 billion tons of plastics have been produced by humans since 1950 (29), while our annual production of plastic in 2020 was 367 million tons (30).

2. 3D printing biopolymers The advent of 3D printing technologies has revolutionized the design and rapid prototyping for the digital fabrication of physical objects (31). The programmable patterning of resins, powders, or inks in the 3D space using computer numerically controlled (CNC) stages that are integrated with various solidification processes, has enabled the fabrication of architected materials with a wide range of tunable mechanical, thermal and electrical properties. The specific patterning and solidification process used by a given 3D printing method defines the dynamic range of the feature size resolution and the type of printable materials that the given 3D printing method can span and use, respectively (32). Scientists, engineers, designers, and makers have at their disposal a wide palette of material feedstocks to process. Within the material palette, printable natural-derived biopolymers are gaining considerable attention. Natural-derived feedstocks for 3D printing are enabling the development of biobased architected material solutions that are set to transform our daily lives and entire industries (33). 2.1 Tools and techniques In this section, we focus on describing the process mechanism of 3D printing methods that have been utilized with renewable natural-derived feedstocks for applications other than drug delivery, tissue engineering, and pharmaceuticals. It is widely known that these industries employ implantable, bioresorbable biomaterials that are usually loaded with a cellular or chemical component for therapeutic purposes. Currently, several 3D printing technologies have been developed to construct biopolymer products and structures. For example, light-based 3D printing technologies employ light of a specific wavelength (UV or visible wavelength) to selectively solidify a liquid resin at predefined patterns through a process called photopolymerization. The end-effector, which is responsible for the photopolymerization process is a rastering laser mounted below the liquid resin vat. The illumination process is repeated layer by layer until the 3D physical object is created on a stage. During the process, the stage is submerged in the liquid resin vat and programmatically moves across its vertical axis and away from the end-effector using a linear axis every time a layer is created. The process is called stereolithography (SLA) and newer methods that aim to increase speed and

3D printing with biopolymers: toward a circular economy

feature size resolution have emerged during the last decade. The ones related to higher speed are the digital projection lithography (DLP) method and the continuous liquid interface production (CLIP) method. Both process mechanisms are based on the basic photopolymerization concept of SLA due to the employment of end-effectors that allow illumination and solidification of an entire layer, as opposed to the point laser source employed by SLA. Direct ink writing (DIW) is another example in the group of extrusion-based 3D printing technologies. It uses an end-effector that is loaded with an viscoelastic ink. The rheological properties of such inks allow them while being heated or cooled, to flow through small needle gauges and maintain their shape fidelity when deposited on a print bed under ambient conditions. The print bed is usually mounted on a CNC translational stage with moving degrees of freedom in the x-y cartesian plane. Users extrude such inks on a programmable translational stage in a continuous filament form using a mechanical syringe pump or a pneumatic regulator. Fused deposition modeling (FDM) is also a well-known method in the category of extrusion technology. Both methods can be applied for the printing of thermoplastic polymer that are either loaded in pelletized form within a temperature-controlled syringe or are directly fed in filament form through a temperature-controlled nozzle with a heat brake. In both cases, the deposited material solidify directly after extrusion using cooling below their glass transition temperature, as they experience ambient conditions. Extrusion-based technologies allow printing of a broader range of biopolymers compared to light-based technologies with most of the relevant reported literature occupied by printable biopolymers using DIW and FDM. 2.2 3D printable biopolymers 3D printable biopolymers that have been demonstrated in the context of light-based 3D printing technologies are significantly smaller compared to the ones that have been reported in the context of extrusion-based 3D printing technologies. Sustainable photocurable resins have been developed for SLA and DLP processing using renewable feedstocks (from lignocellulosic biomass, oils, and animal products) or waste feedstocks (e.g., waste cooking oil) (34). Motivated by the abundance of lignocellulosic materials, Melilli et al. developed a methacrylated carboxymethyl cellulose-based ink for DLP printing (35). The hydrogel exhibited extremely promising mechanical and swelling properties. Skliutas et al. used a single biobased resin derived from soybean and demonstrated that the resin can be processed without the addition of a photoinitiator under visible light wavelength (36). The study showed that the resin can be employed with a benchtop DLP 3D printer fabricating 3D objects with reproducible micron-scale feature resolution. In another interesting study, Barkane et al. demonstrated that an acrylate epoxidized soybean oil can be used as a resin for SLA printing under UV light (37). The authors studied in detail the photopolymerization response of the vegetable oil-based formulation as a function of

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the photoinitiator and the UV curing time and demonstrated its excellent printability with potential scale up for commercial applications. The pool of 3D printable biopolymer inks that have been demonstrated for extrusion-based includes a wide variety of composite biopolymer inks with remarkable properties (33). Inks for DIW processing include lignocellulose, starch, algae, and chitosan-based biopolymers. Motivated by the abundance of hemicellulose in nature and its underutilization in the 3D printing field, Gocke et al. devised a formulation strategy for DIW that will not require complex chemical modifications widely reported for cellulose-based inks (38). To accomplish that, the authors extracted hemicellulosic polymers from lignocellulosic agricultural wastes (corn cobs) and demonstrated that can be 3D printed without any chemical modification or blending to achieve favorable rheological properties (39). Lignin is another material system that will play an important role in the transition to advanced materials engineering for sustainability due to its availability on an industrial scale (40). Many ink formulation strategies for DIW have been reported in the literature using lignin as the base material system since it allows: (a) processing at room temperature, well below the degradation temperature of lignin around 200
C, and (b) shear thinning properties during extrusion and stress recovery for shape retention upon deposition on the print bed. Ebers et al. demonstrated a fully bio-based liquid crystalline lignin/hydroxypropyl cellulose aqueous inks and a detailed study to identify the printability window as a function of different ratios between the composite ink constituents (40). In another study, Jiang et al. reported a low-cost lignin-based ink formulation that overcomes the challenges of weak mechanical properties of printed parts after solvent evaporation due to the hydrophilic nature. The authors demonstrated superior mechanical properties of the lignin-based printed ink compared to printed cellulose, as well as stability in water, under heat, and UV-blocking performance (41). Another group of natural biopolymers extracted from the marine biomass and suitable for DIW upon blending with appropriate cross-linking agents and other prep-polymer solutions are algae-based materials such as alginate, agarose, and carrageenan. These materials are widely used for research purposes in the 3D bioprinting field for tissue engineering and drug delivery application (42e44). Lastly, Sanandiya et al. used the flies’ chitin to develop a chitosancellulose biocomposite ink, quoted as fungal-like adhesive material (FLAM). The composite showed characteristics similar to rigid polymer foams, metal foams, and softwood and the authors showed 3D printing of FLAM objects using a large-scale FDM process (see Fig. 13.1) (45). 2.3 Challenges and opportunities Key challenges related to 3D printing technologies with biopolymers are the combination of reproducible printability with the slow building speed and weak mechanical properties of printed end parts. Starting with the challenge of printability, biopolymers cannot be utilized in their native forms, and time-consuming efforts are being made to tailor

3D printing with biopolymers: toward a circular economy

Fig. 13.1 Additive manufacture of fungal-like adhesive materials.

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them into valuable 3D printing feedstocks mostly through physical blending with other biopolymers and green additives. Specifically, the concentration, molecular weight, and composition of soft biopolymers, directly determine the viscosity, gelation mechanism, and mechanical properties of the final gel (46). The material parameters in combination with the process parameters, such as nozzle diameter, extrusion rate, and stage speed influence the printing fidelity, build speed, resolution, and most importantly functional performance of the end part depending on the application. It is widely known that being able to match mechanical and thermal performance characteristics of petrochemicalbased polymers such as thermoplastic, thermosets, and elastomers, with biopolymer formulations, is a challenge that remains to be solved. Turning 3D printing with biopolymers into a viable manufacturing process that can provide us with sustainable infrastructure for our daily lives, requires the automated synthesis and characterization of tailored biopolymer formulation inks and structures, the development of novel green chemistry solvent systems to improve rheological properties, the modification of commercial printers with advanced real-time process control technologies, and further development of various post-processing technologies, such as surface coating and plasma irradiation, to obtain and maintain the desired shape of printed products. Particularly, the chemical modification of biopolymers and physical blending of different types of biopolymers with other ingredients driven by automated physical and computational experimentation systems can be a favorable strategy to formulate products with favorable processability, printability, mechanics, and bioactivity in an accelerated manner.

3. Material innovation: 3D printing biopolymer performance 3D printing is opening new horizons in manipulating nano, micro, meso, and macroscale structures for material performance. To explore the growing demand for regenerative biocompatible materials in the development of localized macro-scale AM parts, we must first explore the complex hierarchical molecular structure of biopolymer systems and the organism from which they are derived. Naturally occurring polymers such as cellulose, chitin, starch, sodium alginate, and pectin exhibit remarkable functional and structural properties making these biopolymers ideal candidates to replace their petrochemical-derived synthetic counterparts (47,48). Here, we focus on the extensive role of chitin and cellulose in the acceleration of biopolymer 3D printed performance, alongside assessing the inclusion and impact of fundamental biocompatible structural reinforcers (49e51). While deacetylated chitin or chitosan is most prevalent in the exoskeleton of crustaceans (52), surplus shellfish and their chitin containing byproducts may not be readily available across all common waste urban environments (51,53). Therefore, as an alternative means of cultivation, chitin can also be extracted through the valorization of food

3D printing with biopolymers: toward a circular economy

waste utilizing black soldier flies. During reproduction, black soldier flies generate larvae. The larvae are a low-cost feedstock and are relatively under-utilized as a ubiquitous source of chitin (53,54). Conversely, as cellulose is the most abundant of all naturally occurring organic compounds it is commonly found in most geographic regions. For instance, cellulose accounts for 90% of cotton and 50% of wood fibers. As such, it is an organic component that can be recovered from recycled fibers (50). Hence, chitincellulose biopolymeric compounds have the capacity to be locally produced and can degrade without compromising indigenous ecosystems (55). As nature’s most abundant biopolymers, structural polysaccharides cellulose and chitin exhibit extraordinarily diverse mechanical properties (56). Despite their chemical structures being closely related and both occurring as a structural constituent within most eukaryota and bacteria environments, they rarely exist within the same organisms (57,58). The exception occurs, however, within a class of eukaryota organisms known as oomycetes, which grow in mycelial form as fungi. Uniquely, these nonhomogeneous organisms are predominantly composed of cellulose rather than typically defined by their chitinous content, as in the case of fungi. Research into the pathogenic nature of some oomycetes has led to integral studies of potential biosynthetic routes for disease control. Consequently, the investigation of these biopolymers into micro scale medicinal or pharmaceutical applications has broadened the spectrum of bioinspired material development (49,50). Due to the broad locality of sources from which chitin and cellulose can be derived, in addition to their unique molecular affinity, research surrounding the introduction of small quantities of chitosan to cellulose fibers has been explored in the context of large-scale 3D printed manufacturing, notably documented in the study of Fungal-Like adhesive material (FLAM) (45,53). Spurred by the notion of generating bioinspired materials which are not required to combine with synthetic plastics through the copious use of strong and pollutant acids, chitosan can be produced through the deacetylation of chitin utilizing acetic acid (4%e10% table vinegar) to allow effective binding with the relatively chemically stable cellulose (45). Through optimum formulation ratio analysis, chitosan-cellulose FLAM composites of a 1:8 ratio (w/w) were reported. From a supply chain perspective, the cost to develop these materials would reside in a region similar to commodity plastics and would be much lower than the commonly exploited PLA or ABS in 3D printing applications specifically (58). Furthermore, the reported mechanical properties of FLAM are within the range of analogous cellulose composites, such as medium to low-density woods or high-density foams. However, it is worth considering that FLAM is a direct simulation of natural materials which are synthesized on the microscale such as those observed within the oomycete wall. The closest comparative mechanical properties to FLAM are observed in bespoke rigid polyurethane foam commonly utilized in reconstructed bone scaffolds (45).

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The unique characteristics of FLAM offer the potential to adapt and develop largescale 3D printing technology. In this study a bespoke design-to-manufacture program was developed which eliminates the historic complications associated with the dependence on hazardous solvents as part of the development process, ultimately leading to inconsistent cellulosic 3D printed forms (59). Consequently, the discovery of FLAM has accelerated the potential to manufacture tunable 3D printed materials which can accommodate large-scale applications. Furthermore, the robust nature of FLAM allows the exceptional bioinspired material to undergo rendering/fabrication processes such as sanding, casting, sawing, and molding in addition to 3D printing. Additionally, FLAM is entirely biodegradable, lightweight, and made from two of the most readily accessible biopolymers on earth. The unique utility and accessibility of this resource will encourage more regionally specific bio-inspired manufacturing policies and governing approaches (45,53). The role of water in the development of biocompatible systems has also been explored extensively in the design and manufacture of micro-to-macro scale 3D printing composites (60,61). In nature, water is integral to all biological systems by enabling tunable structural interactions between molecules. For instance, the presence of water within chitin provides essential flexibility. Hence, the physical properties within these biological material systems can be altered according to the degree of water content (62). By exploiting the rheological composition of biodegradable hydrogel composites and aggregate concentrations, a nozzle-incorporated extrusion system can provide a feasible route to AM technology without the required casting molds needed in conventional manufacturing techniques. The resulting large-scale bioplastic compositions exhibit structural properties and functionalities ranging from the micro to the macro scale (47). This is comparable to those chitosan forms which employ traditional AM processes observed through the utility of injection casting into epoxy resin molds to make industrial products such as drinkable cups and container pots (45,49,53). Hence, the utility of water-based nozzle or robotic digital fabrication has been investigated to demonstrate the capabilities of alginate and chitosan-based biosystems for selfsupporting, biodegradable macro-scale 3D printed scaffolds (47). The actuated extrusion system approach to the AM process of bioinspired material systems has given precedence to the construction of sophisticated hierarchical water-based structures which can be degraded in closed-loop regional systems (47). Further research into multi-biopolymer composites with tunable functionality for macro 3D printing has also been documented, as an extension of water-based digital fabrication research (61). Comparatively, a considerable selection of non-toxic, biodegradable, biopolymer derivatives were formulated based on their inherent physical and mechanical properties. This research ultimately will have a large influence on the steps toward fabrication information modeling (FIM) (47), whereby 3D composite materials influenced by their atomistic properties can determine their digital manufacturing output (61).

3D printing with biopolymers: toward a circular economy

The introduction of inorganic and organic additives to a structural matrix of chitosancellulose blends to produce novel bio-cement composites were further reported (61). Aimed at creating architectural-scale structures, blends are composed of chitosan, cellulose, starch, pectin, and calcium carbonate in varying proportions, influencing the degree to which the structural, mechanical, and optical properties can be regulated. By varying specific chemical concentrations to alter the hierarchical functionality of each biopolymer it is possible to apply tunable mechanisms to the novel biocement using the water-based digital fabrication platform for applications from the nano to macro scale. This programmable tunability will allow the physical, optical, and mechanical properties of the biodegradable bio-cement to be controlled, providing bespoke regional material specifications. These investigative studies have paved the way for novel bioinspired materials with mechanical properties that span from nano to macro manufacturing capabilities. Although these technologies are in their relative infancy, the potential to utilize these resources opens the possibility to transition away from reliance on petroleum-based plastics.

4. Process: life-cycle analysis of biopolymers and 3D printing process Life cycle assessment (LCA) is an approach that seeks to comprehensively assess the environmental impact of products or systems. While environmental impact remains the core focus, economic and social impacts have also been integrated. Since its early development in the 1960 and 1970s, it has evolved to encompass standardized methods including ISO standards 14040/14044 (63). Its defining characteristics remain the use of a life cycle perspective, where all the processes required to produce a product are considered and can be compared in terms of environmental impact, its inclusion of a broad set of environmental impacts, from climate change to eutrophication to toxicity and human health, its quantitative approach to measuring impact, and its core foundation in the natural sciences (64). Beyond a core focus on product systems, LCA is used to assess the impact of cities and infrastructure, companies and energy, transport, and waste management systems (63). Here we review LCA studies of biopolymers and biomaterials, before considering LCAs of 3D printing processes. We then review the select few studies that have looked at the intersection of these areas using LCA. While important work is being done in this area, there are many complexities in undertaking LCAs related to 3D printing processes and products. Complex factors include material development and characterization, process control and modeling, and the characteristics of machines, tools, and software used (65). 4.1 LCA of biopolymer production Biopolymer-based materials can be understood as a type of bioproduct based on biomass feedstock. Biomass contains biogenic carbon or carbon that is taken up and stored by

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plants during their life cycle. Thus, biopolymer-based materials are often viewed as being “carbon neutral”, although the timing of carbon uptake, storage and release is of critical importance to understanding their impact on climate change (66). This idea of carbon neutrality is central to LCAs comparing biopolymer-based materials to petrochemical materials on the dimension of climate impact. In LCA studies, biopolymers extracted from first-generation biomass, or biomass that competes for arable land for food and feed (i.e., corn, oil, and sugar crops) (67), have registered high levels of carbon emissions from land-use change and other impacts such as eutrophication due to their reliance on industrial agriculture, showing the importance of setting system boundaries in LCA studies to account for impacts throughout the value chain (66). The use of second-generation biomass, often defined as lignocellulosic byproducts such as agricultural residues, has been shown to reduce environmental impact. For example, in studying three alternative polyhydroxyalkanoates (PHAs) made from crude vegetable oil, biodiesel byproduct, and used vegetable oil, it was found that the impact of the processing stage primarily stemmed from the resource used and used oil had a significantly lower environmental impact than virgin oil (68). The use of third generation biomass, often defined as relating to algae and micro algae-based feedstock and food waste (66,69), has been found to reduce LCA impact “hot spots”, including land-use, acidification, and eutrophication, when used as an alternative feedstock for producing conventional bioplastics such as PLA (70). See Fig. 13.2 for an overview of first, second, and third generation biomass (69).

Fig. 13.2 Generations of biomass.

3D printing with biopolymers: toward a circular economy

Likewise, it is important to account for different end-of-life disposal options in biopolymer-based materials, such as recycling, incineration, home and industrial composting, and anaerobic digestion. Disposal options can have more or less impact depending on the material, soil type, efficiency of the technologies used, and policy environments including the use of carbon credit schemes (71,72). For example, an LCA study of common commercial bioplastics, polylactic acid (PLA), thermoplastic starch (TPS), bio-polyethylene (bio-PE), and bio-polyethylene terephthalate (bioPET), found that recycling has a large reduction in impact by substituting for the virgin product, with composting and transport also being significant factors (72). Other studies have concluded that the energy intensity of production processes associated with more novel biopolymers can counterbalance environmental benefits from biogenic carbon storage; as such, process efficiencies and scale-up are needed to reduce energy intensity (73). For instance, a promising study of various production scenarios for bioplastic feedstock produced in an algae biorefinery demonstrates reductions in carbon emissions between 67% and 116% compared to a petroleum-based plastic feedstock (9). 4.2 LCA of 3D printing biopolymers In an increasing number of studies, LCA has been used to assess the impact of 3D printing technologies. At the manufacturing systems level, studies show how a distributed manufacturing system, enabled by 3D printing, can have significant energy efficiency savings compared to centralized production on a per unit basis (74,75). Similarly, Kreiger et al. (76) found that distributed recycling of high-density polyethylene (HDPE) versus centralized recycling had a lower environmental impact, where the use of distributed recycling could be coupled to distributed 3D printing manufacturing. Looking at manufacturing process comparisons, lower environmental impact from material savings that can come from using a 3D printing process over conventional manufacturing methods has been demonstrated using LCA methods in concrete construction (77). In the 3D printed construction of concrete pillars, lower environmental impact was particularly associated with the building of a single pillar; 3D printing becomes comparable with conventional methods when multiple pillars are built using a reusable mold (78). The environmental impact of 3D printing has been compared to CNC machining (79), and metal AM with traditional casting, extrusion, rolling, forging, and wire drawing (80). The comparative impact of powder, printer, and power usage was studied for selective laser melting (81). In most studies, energy usage is found to be a dominant factor in the overall environmental impact. Other significant factors include spatial and temporal utilization (82) and material use (77,78,82,83). As noted by Faludi et al. (83,84), energy usage is often determined by material choice; for instance, thermoplastics require heated extrusion. In this way, the choice of materials is deeply implicated in the energy usage and therefore the environmental impact of the 3D printing process.

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Fig. 13.3 Embodied energy per part, 3D printing mica material versus ABS.

LCA studies of biopolymer-based 3D printing are limited, offering an opportunity to advance this specific research area. Examples include the study of the environmental impact of closed-loop recycling of PLA extruded by an FDM 3D printer in continuous cycles (85). These accord with other studies of the positive impact of recycling biopolymers compared to industrial composting or incineration (72). Faludi et al. (83) developed a series of compostable 3D printable biomaterials, using the filler ingredients of saw dust, orange peel, nanocellulose, powdered mica, binders such as sodium silicate, polyvinyl alcohol, pine rosin, and other ingredients including rice flour, gelatin, wheat dextrin, and glycerin. Materials were specifically selected for low print energy, low toxicity, and biodegradability. The study conducted a comparative LCA of the materials and 3D printing process, comparing results to 3D printing ABS. They found that printing energy was reduced by 75% (see Fig. 13.3), and embodied impacts of materials were reduced by 82%, reducing the overall impact per part printed by 78% (83,86) This study demonstrates the potential scale of environmental impact reduction that can be achieved when 3D printing with biopolymers. Further research is needed to develop LCA studies for other biopolymer-based 3D printing materials, combining print process impact with embodied material impact (including biomass sourcing) with various end-of-life scenarios.

5. Material supply chain: sourcing biopolymers for local production The potential alignment between 3D printing and the circular economy may also stem from disruption at the level of the manufacturing supply chain. Being software-defined (87), 3D printing reduces the capital required to reach a minimum efficient scale of

3D printing with biopolymers: toward a circular economy

production, replacing the economics of economies-of-scale production with economies of one, given the production of highly customizable, 3D printable products (88). This new economics can lower barriers to entry, allowing production to collocate with demand, enabling distributed, local production (89). With the potential for mass personalization and localized, flexible production of goods close to consumer demand, 3D printing has been investigated as a technology that enables the redistribution of manufacturing (90). Redistributed manufacturing is understood as “Technologies, systems and strategies that change the economics and organization of manufacturing, particularly with regard to location” (91). This logic has led authors to investigate the potential of redistributed manufacturing when it comes to materials supply, using waste plastic for 3D printing as an opportunity to investigate whether materials for manufacturing could be sourced locally, reducing emissions from transport costs, and growing capacity for local cycling (20,22,92). Here, we broaden this perspective by considering how sourcing biopolymers for 3D printing could be part of a redistributed manufacturing future and thus align the technology with circular economy aims. We further examine the challenges of sourcing biopolymer feedstock for 3D printing. 5.1 Sourcing biopolymers Relying on biomass for centralized mass manufacturing is difficult given that biomass is heterogeneous, and costly to transport and store. Petrochemical feedstock extraction and processing need not be geographically proximate given that compositionally, petrochemicals are relatively stable and homogeneous, making them easy to transport and store over long distances (93). This means that one of the main benefits of sourcing biopolymers for 3D printing is that the distributed nature of biopolymer feedstock is in accordance with the logic of distributed digital fabrication. At local scales, biomaterials can be harvested from a diversity of terrestrial and marine habitats. The production of ingredients for biomaterials can also take place in artificial environments such as the cultivation of microalgae to produce oils, proteins, and carbohydrates as building blocks for bioplastics (94). Biowaste from the byproducts of agricultural, forestry, and aquaculture systems also has large potential. For example, biowaste from the shellfish industry can be valorized by extracting the biopolymer chitin and its derivative chitosan for making gel, film, and fiber materials (50,95,96). Moving away from an extractive resource paradigm, where primary resources are extracted in mass quantities, processed in linear supply chains, and sold to distant consumers, local biopolymer production may open opportunities for materials to be produced, consumed, and returned as nutrients within specific ecosystems and coupled social-ecological systems such as agricultural environments. The benefits of this approach can be seen in regenerative agricultural practices where valuable nutrients are being returned to cropland in the form of human waste from surrounding urban environments

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(Fig. 13.4) (97). If biopolymers for 3D printing were locally processed and distributed, nutrients could be returned to areas of biomass production after the product’s end of life.

Fig. 13.4 Nutrient return in mainstream versus regenerative agriculture (97).

3D printing with biopolymers: toward a circular economy

5.2 Challenges The first challenge of note is the inherent heterogeneity of biomass feedstock used to source 3D printable biopolymers. The local abundance and distribution of biomaterials remain highly sensitive to variation in the biome and even ecoregional conditions (93,98). This makes it challenging to source feedstock across distributed geographical areas. Moreover, chemical and physical heterogeneity in biomolecules from species make it difficult to develop a consistent feedstock in terms of material properties. Biopolymer yields across different species varies, in addition to variation in chemical and physical features. Concerning lignocellulosic biomass, for example, the composition and structure of cellulose, hemicellulose, and lignin varies, as well as physical features including accessible biomass surface area, cellulose crystallinity, and degree of polymerization (99). These features interact with the use of different extraction methods, leading to a large diversity of feedstock characteristics as input for 3D printable biopolymer feedstock. Such heterogeneity needs to be quantified and understood to develop reliable feedstock for 3D printing. The second challenge relates to the processing of biopolymers. Today, commercial bioplastics are mainly derived from corn (to make PLA), sugar cane (to make bio-PE), and waste fats and oils (to make second-generation BioPE) (100). Many of these bioplastics are non-biodegradable; for instance, BioPE is made where chemical components are “dropped-in” to existing petrochemical production processes. This compromises the biodegradability of the resulting plastic, illustrating that it is the chemical structure and not the origin of ingredients that determines biodegradability (99). Third, there is the need to develop appropriate sourcing practices that do not risk over-extracting source ecosystems or causing unintentional environmental damage. Relying on primary biomass for harvesting biopolymers, such as corn for making PLA, risks competition for arable land for food, water stress, and carbon emissions from land-use change (67). Biomass sources that do not incur land-use change emissions nor compete with food production include algae and unavoidable food waste. A promising alternative is the use of algae biomass for 3D printing, given their fast growth rate, and tolerance to varying environmental conditions that allow them to be cultivated in areas not needed for food (101,102). Green microalgae and cyanobacteria have been used to produce a range of biodegradable plastics through the use of biorefineries (9e102), (103,104), while macroalgae, or seaweed, can be cultivated in offshore environments including open ocean and offshore wind farms, as well as onshore coastal environments (105). To illustrate the promise of waste biomass, waste from the shellfish industry can be valorized by extracting the biopolymer chitin and its derivative chitosan for making gel, film, and fiber materials (49,95,96). Chitin has also been harvested from black soldier flies fed on organic food waste, demonstrating a viable bioconversion of urban waste into

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materials for sustainable manufacturing (53), and the potential to produce at distributed local/regional scales (106). Food waste is also being researched as a viable biomass source for PHAs (107), helping to bring down the cost of production (108). Specific biopolymers can be isolated from individual organic waste streams for bioplastic synthesis. For example, suberin from cork powder and bark (109), gelatin from slaughterhouse waste, pectin from citrus peel, cutin from tomato peels, and cellulose from parsley and spinach stems, rice hulls, and cocoa pod husks (110,111). Even if more regenerative sources are used, effective governance is required in the context of increased use. If the sourcing and cycling of biomaterials is not accompanied by effective governance at the appropriate scale, there is a clear danger of accelerating the overharvesting of natural resource stocks and undermining biodiversity and ecosystem services (6).

6. Case study: sourcing chitosan from waste The following case study documents the sourcing of chitosan from crustacean waste in the community of Puerto Williams, Chile. As chitosan is one of the most common biopolymers used in 3D printing, this case study illustrates the challenges and opportunities of localizing biomass sourcing. As discussed in section five, localizing biomass sourcing for local manufacturing is fundamental to realizing the potential of 3D printing biopolymers for a circular economy. 6.1 Overview Puerto Williams is a city located on Navarino Island next to the Beagle Channel and is the southernmost city in the world (112). One of the main commercial activities of the island is fishing, mainly focused on the Chilean king crab. Being only accessible by sea or air (113), waste management is difficult, an issue accentuated every year during the king crab fishing season, where several tons of exoskeleton are dumped in a local landfill. The crab consists of 20% edible meat. The remaining 80% is inedible (114), most of which is the discarded exoskeleton of the crab. Moreover, the spider crab exoskeleton is composed of around 17% chitin (115), a biopolymer that has great potential for digital manufacturing due to its material properties (49,116). This raises the opportunity of using local biomass that is considered an undesired waste as a resource for local manufacturing. Given the utility of chitosan as an attractive 3D printing material (45,60), locally extracting chitin has the potential to promote a more regenerative and circular supply chain for 3D printing biopolymers. Fig. 13.5 provides an overview of the process undertaken from biomass collection, extraction, and fabrication.

3D printing with biopolymers: toward a circular economy

Fig. 13.5 Chitin-based material production.

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6.1.1 Extraction and fabrication 6.1.1.1 Chitin and chitosan extraction For the selection of the local chitin extraction method, sustainable extraction methods from the literature were compared with the feasibility of being replicated with local experience and tools. To extract chitin, the crab exoskeleton needs to go through the phases of demineralization, deproteinization, and depigmentation. To obtain chitosan, a further deacetylation process is needed (96). The traditional process for chitin extraction is highly energy-intensive and requires strong chemicals such as NaOH and HCl (115,117). To develop a more sustainable process, other methods were researched that used fewer chemicals, introduced biological elements, or had a more efficient use of energy by changing the technology or mechanisms used for extraction. A fermentation method using lactic acid was selected, due to its ease of replication in contexts with little access to advanced laboratory technology, its low energy use, and it is zero use of chemicals that may impact surrounding ecosystems (117e120). The fermentation method chosen was based on the research of Benavente et al. (119) which used lactic acid from whey. The study added a further chemical step to obtain chitosan as lactic acid was not enough to reach deacetylation. Given the constraints of locally available technology, extraction was carried out only with lactic acid. 6.1.1.2 Fermentation The crab exoskeleton was cleaned, dried, and ground. In addition, lactic acid was prepared using milk and lemon, following the steps outlined by Benavente (119). In accordance with the literature, to promote an ideal fermentation in the lactic acid, a pH level of 6.0 was maintained, with a constant temperature between 30 and 40
C. The solution of lactic acid and crab exoskeleton powder was then left to ferment for 9e10 days in an anaerobic medium at around 30
C, being shaken three times a day. Tools were used to support the process including a dehydrator and a hot surface to maintain a constant temperature. Temperature gauges were used to monitor the process. 6.1.1.3 Fabrication After the fermentation process, the solution was transferred to a saucepan, adding glycerin and vinegar at approximately 70
C and stirring for about 5 min. To cast a biopolymer film, the solution was poured into a silicone mold and left to dry in the dehydrator at 40
C for 48 h. A dark orange semi-transparent film was created with a slightly sticky surface. The sample was flexible and could be machined with laser cutting and manual techniques. 6.1.2 Discussion and future potential studies This study shows the potential of sourcing local biomass waste to develop biopolymers for local manufacturing. This accords with previous research (45,47) showcasing the

3D printing with biopolymers: toward a circular economy

potential of chitin combined with digital fabrication for material innovation. While further research is needed into 3D printing the solutions obtained, this study offers promise for developing locally sourced materials that can be integrated with 3D printing technology in remote manufacturing contexts. The project was successful in terms of technical development, being able to fabricate sheets from waste crab exoskeleton. The following opportunities and challenges were identified. In terms of future research opportunities, at the regional level, it would be advantageous to secure large volumes of the same type of biomass (93). Given the seasonality of crab harvesting, appropriate management and storage would be necessary. To avoid issues of over-extraction, diversifying chitin extraction from other crustaceans would be important, in addition to fungi or local insects. A comprehensive analysis using LCA would help understand the environmental impact of the whole life cycle of chitinbased fabrication, including material extraction, biodegradation, and the quantity and type of energy demands (121). The integration of 3D printing could explore a greater breadth of material performance, including the mechanical properties of chitin-based materials. Further collaboration at the local level with academic research centers and indigenous communities would enrich understanding of the scope and context of further material exploration. Several challenges were identified at the technical level. More research is needed on the extraction process to facilitate the fabrication of materials. In this regard, other acidic fermentation media could be experimented with, in addition to more complex chemicals that can be maintained in a closed process loop to reduce their impact on the surrounding ecosystem. Further research is also needed to achieve a consistent material suitable for 3D printing, taking into account how characteristics like viscosity, density, and solution concentration impact the quality of the print. A further challenge at the social level was identified, that of the need to integrate knowledge from diverse areas such as biology, chemistry, engineering, design, ecology, and material design. The complexities of integrating these knowledge sets into a clear methodology was especially challenging given the need to communicate it to nonspecialized people or professionals in the community. Further work is needed to develop methodologies for local materials production and fabrication in collaboration with local, contextual community knowledge.

7. Conclusion and future trends At the material innovation level, more research is needed in exploring the performance that can be achieved by adding structure via 3D printing to biopolymer building blocks. Research is advancing on new biopolymer-based materials for high-performance applications, such as self-healing materials at the nanoscale (122), artificial silks from protein building blocks (123), mussel-inspired underwater adhesives (124), super hydrophobic

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surfaces inspired by the lotus leaf (125), and reversible adhesives inspired by the gecko (126). Researchers are beginning to build bridges between diverse fields of study, bringing advanced 3D printing methods and biopolymers together, such as in the production of architectural-scale structures from chitosan using water as a programmable building block (116). However, much more is needed to develop advanced biopolymer 3D printing techniques that can approximate nature using simple molecular building blocks to create high-performance materials. Current applications of 3D printed biopolymers are predominantly in the biomedical field, including tissue engineering, scaffolds, organs, and drug delivery (42,43,127), as well as pharmaceutical and food applications (127). Motivated by sustainability interests, 3D printing biopolymers are being explored for architecture and load-bearing structures (45,49). Additional emerging applications for 3D printing biopolymers include wastewater treatment, electronic circuits, and wearable sensors (127). At the process level, more studies are needed on the life-cycle impact of a range of printable biopolymers, considering extraction and biopolymer processing, as well as printing and end-of-life disposal. For example, to better evaluate LCA hotspots of energy demand and global warming for biopolymers from algae, research needs to account for changes in the efficiency of extraction and processing technology through technology improvements and upscaling as biopolymer production moves beyond the lab scale (70). To provide a more holistic picture of 3D printing biopolymers, LCA studies of biopolymer production need to be combined with 3D printing process LCAs, and end-of-life disposal options. Moreover, there is an opportunity to investigate the recycling of 3D printed biopolymer-based parts, through processes of depolymerization and repolymerization (128). At the level of biopolymer production, more research is needed into the potential for 3D printing with biopolymers to increase participation in the circular economy. This aligns with recent scholarship that has identified the need for increased focus on the social and institutional dimensions of circular economy practice (129). As global trends in mass customization (130), digital fabrication (131), open design (132) and (re)distributed manufacturing (91) begin to transform how, and by whom, products are manufactured, entrepreneurs are showing that this is possible within biopolymer-based production (133). More research is needed into whether 3D printing biopolymers will be part of the wider democratization of technological practices - namely, the rise of open access fabrication labs and the communities who use them to share materials, tools, technologies, and knowledge with like-minded peers (134). Finally, if 3D printing biopolymers is to contribute to ecological regeneration at the supply chain systems level, qualifying this position and the scope of its validity will require an analysis of nutrient flows for a variety of local case studies. Important variables include the rates of extraction of nutrients for biomaterial feedstocks, the rates of return for nutrients after a product’s end of life, and their respective sustainable boundaries (e.g.,

3D printing with biopolymers: toward a circular economy

enabling nutrient replenishment in soils and avoiding eutrophication of oceans), and considering ecosystems’ interdependencies. Evaluating the conditions in which ecological benefits are maximized and the risks of environmental degradation are minimized must consider ecosystem services, and social-ecological systems and their resilience (6,7,135,136,137,138).

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399

Index Note: ‘Page numbers followed by “f ” indicate figures and “t” indicate tables.’

A

Action of flame retardants, 103e111 Additive manufacturing (AM), 135, 137e145, 157e158 biopolymer crystallization, 149e150, 150f biopolymers, 1e3, 2f challenges and future trends, 7e9 classification, 5e7, 5f fabricated biopolymer composites, 146e148, 147f filler alignment, 150e151, 151f filler homogeneity, 151e152 interfacial bonding, 152 interlayer bonding, 152 manipulation of material properties by, 148e152, 149f material extrusion, 139e141, 139fe140f techniques and principles, 138e142, 138f vat polymerization, 142, 142f Additive Manufacturing Technologies (AMT), 258 Agarose, 275 Alginate, 56, 276e277 Automation, 258

B Barrel tumbling, 251 Bi/multi-layer structural design, 198 Binder jetting (BJ), 5, 27 Biobased poly(ethylene terephthalate) (bio-PET), 1e2 Biobased polymer blends, 119e128 Biopolymer-based hydrogels, 3D printing of biopolymers, 65e67, 66f data availability statement, 94 extrusion-based 3D printing of biopolymer hydrogels, 71e85 oscillatory shear rheology, 79e80 other aspects, 80 principle of, 71e73, 73f printability evaluation for, 74e80, 74fe75f steady shear rheology, 76e79, 77fe78f future perspectives, 91e93, 92t

inkjet 3D printing, 85e87 biopolymer hydrogel materials, 86e87, 87f principle of, 85e86 laser-mediated 3D printing, 88e91 biopolymer hydrogel materials, 89e91, 90f principle of, 88e89, 88f polymer hydrogels, 67e71, 68fe69f solidifying process, 80e85 chemical crosslinking, 83e85, 84f enzymatic crosslinking, 82e83 physical crosslinking, 80e82, 82f Biopolymer composites, 3D printing of additive manufacturing (AM), 137e145, 157e158 biopolymer crystallization, 149e150, 150f fabricated biopolymer composites, 146e148, 147f filler alignment, 150e151, 151f filler homogeneity, 151e152 interfacial bonding, 152 interlayer bonding, 152 manipulation of material properties by, 148e152, 149f material extrusion, 139e141, 139fe140f techniques and principles, 138e142, 138f vat polymerization, 142, 142f applications and case studies, 153e157 hydrogels, 157 thermoplastic PLA-based biocomposites, 153e155, 154f thermoset UV curable biocomposites, 155e157, 156f biopolymer nanocomposite inks, 143e145 cure depth, 145, 145f rheology, 143e144 viscoelasticity, 143e144 cellulose nanocrystals (CNCs), 137 composite materials, 135e136, 136f 3D printed biopolymer nanocomposites, 145e152 hard or stiff phases, 137 polylactic acid (PLA), 137 polymer composites, 136

401

402

Index

Biopolymer crystallization, 149e150, 150f Biopolymer hydrogel materials, 86e87, 87f, 89e91, 90f Biopolymer nanocomposite inks, 143e145 cure depth, 145, 145f rheology, 143e144 viscoelasticity, 143e144 Biopolymers, 1e3, 2f, 65e67, 66f additive manufacturing (AM), 3e5, 4f challenges and future trends, 7e9 classification, 5e7, 5f poly(lactic acid) (PLA), 40e42, 41fe42f polycaprolactone (PCL), 42e43 polyhydroxyalkanoates (PHAs), 43e46, 43f polyhydroxybutyrate (PHB), 44, 44f poly(3-hydroxybutyrate-co-hydroxy valerate) (PHBV), 45e46, 45f polysaccharides, 52e59 alginate, 56 cellulose, 53e54, 53f chitin and chitosan, 58e59, 58f lignin, 54, 55f pectin, 56e57, 57f starch, 54e55 proteins, 46e52 casein, 52 collagen, 46e47 gelatin, 47, 48f keratin, 50e52, 51f PEA protein, 49, 49f silk protein, 50 soy protein, 48 whey protein, 52 zein, 49e50 vegetable oils, 59 Bioprinting and hybrid biomanufacturing, 28e30, 28t, 29f, 30t Boronic ester bonds, 287e289

C Carrageenan (Cgn), 275e276 Casein, 52 Cellulose, 53e54, 53f Chemical action modes, 106e107, 107f Chemical bonds, 285e289 Chemical composition of bioinks, 283e292 Chitin and chitosan, 58e59, 58f, 274 extraction, 390 Circular economy

biopolymers, 373e374 challenges, 376e378 3D printing biopolymers, 372e378 3D printable biopolymers, 375e376 tools and techniques, 374e375 extraction and fabrication, 390 chitin and chitosan extraction, 390 fabrication, 390 fermentation, 390 future potential studies, 390e391 future trends, 391e393 introduction to, 372 life cycle assessment (LCA), 381e384 biopolymer production, 381e383, 382f 3D printing biopolymers, 383e384, 384f material innovation, 378e381 3D printing biopolymer performance, 378e381 material supply chain challenges, 387e388 sourcing biopolymers, 385e386, 386f opportunities, 376e378 sourcing chitosan from waste, 388e391 Coefficient of thermal expansion (CTE), 180 Collagen, 46e47, 274 Composite materials, 135e136, 136f Computer-aided design (CAD), 191e192

D Data availability statement, 94 Decellularized bioinks, 277e278 Dicyclopentadiene (DCPD), 197 Digital light processing (DLP), 6, 178, 337 Directed energy deposition, 28 Direct ink writing (DIW), 24e25, 24f, 25t, 167 Disulfide bonds, 287 3D printed bio-based polymers, tissue engineering hydrogels biocompatibility, 293e294 biodegradability, 293e294 bioinks, mechanical properties of, 292e293 biopolymer, physiochemical properties and biological response of, 283e294 boronic ester bonds, 287e289 chemical bonds, 285e289 chemical composition of bioinks, 283e292 disulfide bonds, 287 hydrazone bonds, 286 imine bonds, 286

Index

oxime bonds, 286e287 physical interactions, 283e285, 284t digital light processing (DLP)-based bioprinting, 267e268, 269f extrusion-based bioprinting, 268e271, 269fe270f inkjet-based bioprinting, 271 laser-based bioprinting, 271e272, 271f light-based printers, bioinks chemistry in free-radical chain growth polymerization (FRCGP), 289e291, 290f orthogonal step growth polymerization, 291e292 naturally-derived polymers, 272e277 agarose, 275 alginate, 276e277 carrageenan (Cgn), 275e276 chitosan, 274 collagen, 274 decellularized bioinks, 277e278 gelatin, 272, 273t methylcellulose (MC), 274e275 PCL, 280e281 PEG, 278e279 PLA, 281e282 PLGA, 282 PVP, 281 synthetic polymers/hydrogels, 278e282 stereolithography (SLA)-based bioprinting, 266e267 technologies, 266e272 3D printed biopolymers, 145e152, 311e320 polysaccharides-based medical devices, 315e320, 315te316t, 320f protein-based hydrogels, 3D printing of, 312e314 3D printed medical devices, regulation of, 321e323 4D printing, biopolymers applications of, 215e217, 216f challenges, 217 computer-aided design (CAD), 191 4D bioprinting natural polymers, 199e202 synthetic bioinks, 202e204, 203f 4D printing of biomaterials, structural design for, 198 bi/multi-layer structural design, 198 programmed patterned design, 198

4D transformation, stimuli responsible for, 206e209 biological stimuli, 208e209 chemical stimuli, 208 physical stimuli, 206e208 fabrication techniques, 209e213 extrusion-based bioprinting, 209e210 inkjet bioprinting, 212e213 light-based printing methods, 210e212 stereolithography (SL), 212 future perspective, 217e218 history, 191e192 limitations, 217 mathematical modeling, 197e198 recent advances in, 215 self-actuation, 197 self-assembly, 197 self-folding, 197 self-repair properties, 197 SMP, responses exhibited by, 197 stimuli-responsive materials, 192e196, 192fe195f surface topography, 197 3D printing of biopolymers food printing, 351 material extrusion, 332e333, 332fe333f material jetting, 333e336 inkjet printing, 334e335 laser-assisted printing, 335e336 microvalve printing, 335 tissue engineering, 341e351, 343fe350f vat polymerization, 336e338 digital light processing (DLP) printing, 337 stereolithography (SLA) printing, 336e337 two-photon polymerization (2 PP) printing, 337e338 4D printing of biopolymers biomedical devices, 357, 358f chemical stimuli-responsive materials, 340e341 drug delivery, 354, 355fe356f mechanisms of, 338e341 physical stimuli-responsive materials, 339e340 electro-responsive materials, 339 magnetic-responsive materials, 339e340, 340f moisture-responsive materials, 339 temperature-responsive materials, 339 soft robotics/smart actuators, 357e358, 359fe361f tissue engineering, 351e354

403

404

Index

3D printing techniques additive manufacturing (AM), 11 bioprinting and hybrid biomanufacturing, 28e30, 28t, 29f, 30t Computer-Aided Design (CAD) model, 11e12 future perspectives, 30e31 material extrusion, 23e25 direct ink writing, 24e25, 24f, 25t filament-based, 23 pellet-based, 23e24, 24f material jetting, 19e23 liquid drop jetting, 19e21, 20f melt drop jetting, 21e22 metal drop jetting, 22e23, 22t paste drop jetting, 20f, 21 natural biopolymers, 12e13 other solid-based AM processes, 25e28 binder jetting (BJ), 27 directed energy deposition, 28 powder bed fusion (PBF), 26e27, 26f sheet lamination (SL), 27e28 vat photopolymerization, 13e18 laser beam-based, 13e15, 14f light pattern-based, 15e18, 16f, 18f, 19t 3D printing techniques and biopolymers, 304e310, 305f 4D transformation, stimuli responsible for, 206e209 biological stimuli, 208e209 chemical stimuli, 208 physical stimuli, 206e208

E Electro deposition, 255 Electroplating (EP), 255 Electro-responsive materials, 339 Extraction and fabrication, 390 chitin and chitosan extraction, 390 fabrication, 390 fermentation, 390 future potential studies, 390e391 future trends, 391e393 Extrusion-based bioprinting, 209e210 direct ink writing (DIW), 209e210 fused deposition modeling, 209, 211f Extrusion-based 3D printing, 305e307 biopolymer hydrogels, 71e85 oscillatory shear rheology, 79e80 other aspects, 80

principle of, 71e73, 73f printability evaluation for, 74e80, 74fe75f steady shear rheology, 76e79, 77fe78f

F Fabricated biopolymer composites, 146e148, 147f Fabrication, 390 Fermentation, 390 Filament-based, 23 Filler alignment, 150e151, 151f Filler homogeneity, 151e152 Fire-retardant biopolymers, 3D printing of action of flame retardants, 103e111 biobased polymer blends, 119e128 case studies, 119e128 chemical action modes, 106e107, 107f fire testing, 103e111 flame retardancy, 111e115, 112f, 114fe115f flame retardant system, 108e109, 108f flammability hazard level, 109e111 fused filament fabrication, 116e118, 116f, 117t, 118f materials, 120 mechanisms, 103e111, 103f methods, 120e121, 120t multiwall carbon nanotubes (MWNTs), 107 perspectives, 128e129 physical action modes, 103e106, 104t, 105f poly lactic acid (PLA), 101 results, 121e128, 122f, 124fe126f, 127t synergistic effects, 108e109 Fire testing, 103e111 Flame retardancy, 111e115, 112f, 114fe115f Flame retardant system, 108e109, 108f Flammability hazard level, 109e111 Food printing, 351 Fused deposition modeling (FDM), 167, 198, 231 Fused filament fabrication, 116e118, 116f, 117t, 118f

G Gelatin, 47, 48f, 272, 273t

H Hard/stiff phases, 137 Hydrazone bonds, 286 Hydrogels, 157, 169e171, 169fe171f

Index

I Imine bonds, 286 Inkjet 3D printing, 85e87 biopolymer hydrogel materials, 86e87, 87f principle of, 85e86 Interfacial bonding, 152 Interlayer bonding, 152

K Keratin, 50e52, 51f

L Laser beam-based, 13e15, 14f Laser-induced forward transfer (LIFT), 6 Laser-mediated 3D printing, 88e91 biopolymer hydrogel materials, 89e91, 90f principle of, 88e89, 88f Life cycle assessment (LCA), 381e384 biopolymer production, 381e383, 382f 3D printing biopolymers, 383e384, 384f Light-based printing methods, 210e212 digital light processing (DLP), 211 laser-assisted bioprinting (LAB), 212 Light pattern-based, 15e18, 16f, 18f, 19t Lignin, 54, 55f Liquid drop jetting, 19e21, 20f

M Magnetic-responsive materials, 339e340, 340f Manipulation, material properties, 148e152, 149f Material extrusion (ME), 6, 23e25, 139e141, 139fe140f, 332e333, 332fe333f direct ink writing, 24e25, 24f, 25t filament-based, 23 pellet-based, 23e24, 24f Material jetting (MJ), 5e6, 19e23, 308e309, 333e336 inkjet printing, 334e335 laser-assisted printing, 335e336 liquid drop jetting, 19e21, 20f melt drop jetting, 21e22 metal drop jetting, 22e23, 22t microvalve printing, 335 paste drop jetting, 20f, 21 Materials extrusion (ME), 5 Material supply chain challenges, 387e388 sourcing biopolymers, 385e386, 386f

Media blasting, 250, 250f Medical applications and devices, 3D printed biopolymers for 3D printed biopolymers, 311e320 polysaccharides-based medical devices, 315e320, 315te316t, 320f protein-based hydrogels, 3D printing of, 312e314 3D printed medical devices, regulation of, 321e323 3D printing techniques and biopolymers, 304e310, 305f extrusion-based 3D printing, 305e307 future perspectives, 323e324 material jetting techniques, 308e309 photopolymerization 3D printing technology, 309e310 powder bed fusion methods, 307e308 Melt drop jetting, 21e22 Metal drop jetting, 22e23, 22t Methylcellulose (MC), 274e275 Microplastics, 3e5 Minimally invasive surgery (MIS), 185 Moisture-responsive materials, 339 Multiple stable structures, 181 Multiwall carbon nanotubes (MWNTs), 107

N Natural biopolymers, 6, 12e13, 283, 311e312 Natural materials, 199 N-isopropylacrylamide (NIPAM), 197 Nylon, 172

O Opportunities, 376e378 Oscillatory shear rheology, 79e80

P Paste drop jetting, 20f, 21 PCL, 280e281 PEA protein, 49, 49f Pectin, 56e57, 57f PEG, 278e279 Pellet additive manufacturing (PAM), 6 Pellet-based, 23e24, 24f Photopolymerization 3D printing technology, 309e310 Physical action modes, 103e106, 104t, 105f Physical stimuli-responsive materials, 339e340

405

406

Index

Physical stimuli-responsive materials (Continued) electro-responsive materials, 339 magnetic-responsive materials, 339e340, 340f moisture-responsive materials, 339 temperature-responsive materials, 339 Physical vapor deposition (PVD), 253 Plating, 255e256, 255f PLGA, 282 Poly(caprolactone) (PCL), 1e2 Poly(lactic acid) (PLA), 40e42, 41fe42f, 281e282 Polyamide, 172 Polybutylene adipate terephthalate (PBAT), 2 Polycaprolactone (PCL), 42e43 Polycaprolactone dimethacrylate (PCLDMA), 192e193 Polyhydroxyalkanoates (PHA), 2, 43e46, 43f Polyhydroxybutyrate (PHB), 44, 44f Poly(3-hydroxybutyrate-co-hydroxy valerate) (PHBV), 45e46, 45f Polylactic acid (PLA), 137, 172 Poly lactic acid (PLA), 160 Polymer composites, 136 Polymer hydrogels, 67e71, 68fe69f Polymers, 172e178, 173fe176f, 178f, 180f Polysaccharides, 52e59 alginate, 56 cellulose, 53e54, 53f chitin and chitosan, 58e59, 58f lignin, 54, 55f pectin, 56e57, 57f starch, 54e55 Post-process controls, 234e235 Post-processing methods, 3D printed biopolymers, 230e232 automation, 258 chemical vapor deposition (CVD), 253e254, 254f cleaning post-processes, 240e241 centrifugal force cleaning, 241 solvent washing, 240 ultrasonic bath, 240e241 computer-aided design (CAD), 229e230 controls, 234e235 fused deposition modeling (FDM) filament, 231 future perspectives, 256e259 material, 256e257 mechanical abrasive techniques, 249e251 barrel tumbling, 251

media blasting, 250, 250f vibratory finishing techniques, 251 physical vapor deposition (PVD), 253 plating, 255e256, 255f selective laser sintering (SLS), 231e232 solvent vapor smoothing, 251e253, 252f standards, 257 stereolithography (SLA), 231e232, 231te232t support material, 235e240 post-processing, support structure optimization for, 238e240, 239f powder support, 236e237 solid supports, 237e238 support baths, 238 surface finishing, 246e249 brush and spray coating, 249, 249f gap filling and priming, 248e249 hand sanding, 247e248, 247f surface roughness, 243e246 UV and thermal treatment, 241e243 annealing FDM parts, 243, 244f photopolymers, UV curing of, 241e242 thermal treatment, 243 Powder bed fusion (PBF), 5e6, 26e27, 26f, 307e308 Programmed patterned design, 198 Proteins, 46e52 casein, 52 collagen, 46e47 gelatin, 47, 48f keratin, 50e52, 51f PEA protein, 49, 49f silk protein, 50 soy protein, 48 whey protein, 52 zein, 49e50 PVP, 281

S Selective laser sintering (SLS), 6, 231e232 Shape change effect (SCE), 175 Shape memory alloys (SMAs), 192e193 Shape memory effect (SME), 175 Shape memory hybrid (SMH), 182 Shape memory poly vinyl chloride (SMPVC), 198 Shape-switching biopolymers direct ink writing (DIW), 167 fuse deposition modelling (FDM), 167 typical basic approaches for, 169e178

Index

hydrogels, 169e171, 169fe171f polymers, 172e178, 173fe176f, 178f, 180f typical potential applications, 180e187, 181fe186f Sheet lamination (SL), 5, 27e28 Silk protein, 50 Soft robotics/smart actuators, 357e358, 359fe361f Solidifying process, 80e85 chemical crosslinking, 83e85, 84f enzymatic crosslinking, 82e83 physical crosslinking, 80e82, 82f Sourcing chitosan from waste, 388e391 Soy protein, 48 Starch, 54e55 Steady shear rheology, 76e79, 77fe78f Stereolithography (SLA), 231e232, 266e267, 336e337 Surface folding, 197 Surface roughness, 243e246 Synthetic polymers/hydrogels, 278e282

T Temperature-responsive materials, 339 Thermoplastic PLA-based biocomposites, 153e155, 154f

Thermoset UV curable biocomposites, 155e157, 156f Tissue engineering, 351e354 Two-photon polymerization (2 PP) printing, 337e338 Typical potential applications, 180e187, 181fe186f

V Vat photopolymerization, 13e18 laser beam-based, 13e15, 14f light pattern-based, 15e18, 16f, 18f, 19t Vat polymerization (VP), 5e6, 142, 142f, 336e338 digital light processing (DLP) printing, 337 stereolithography (SLA) printing, 336e337 two-photon polymerization (2 PP) printing, 337e338 Vegetable oils, 59 Viscoelasticity, 143e144

W Whey protein, 52

Z Zein, 49e50

407