Advances in Biomedical Polymers and Composites: Materials and Applications 9780323885249

Advances in Biomedical Polymers and Composites: Materials and Applications is a comprehensive guide to polymers and poly

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Advances in Biomedical Polymers and Composites: Materials and Applications

Table of contents :
Half Title
Advances in Biomedical Polymers and Composites: Materials and Applications
List of contributors
1. Introduction to biomedical polymer and composites
1.1 Introduction
1.2 Classification of polymers and composites
1.3 Fabrication techniques polymer composites
1.3.1 Electrospinning
1.3.2 Melt extrusion
1.3.3 Solution mixing
1.3.4 Latex technology
1.4 Polymers and their composites for biomedical applications
1.4.1 Natural polymers and their composites Collagen Silk Hyaluronic acid Chitosan Cellulose
1.4.2 Synthetic polymers and their composites Polycaprolactone Poly(L-lactic acid) Poly(methyl methacrylate) Poly(lactic-co-glycolic) acid Polyvinylidene fluoride Poly(ethylene glycol)
1.4.3 Gas-permeable polymeric membranes
1.4.4 Other polymeric composites
1.5 Challenges and future trends
1.6 Conclusion
2. Foundation of composites
2.1 Introduction
2.2 Classification of composites
2.3 History of composites
2.3.1 Fiberglass in 20th century
2.3.2 Composite material in our daily life
2.4 Why composites?
2.5 Advantages of composites
2.5.1 Design flexibility
2.5.2 Light weight
2.5.3 High strength
2.5.4 Strength related to weight
2.5.5 Corrosion resistance
2.5.6 High-impact strength
2.5.7 Consolidation of many parts
2.5.8 Dimensional stability
2.5.9 Nonconductive
2.5.10 Nonmagnetic
2.5.11 Radar transparent
2.5.12 Low thermal conductivity
2.5.13 Durable
2.6 Applications of composites
2.6.1 Aerospace/aircrafts
2.6.2 Appliances
2.6.3 Automobile and transportation
2.6.4 Infrastructure
2.6.5 Environmental
2.6.6 Applications of electricity
2.7 Limitation of composites
2.8 Biocomposites and classification
2.8.1 Biomedical composites
2.8.2 Basic requirements and parameters for biomedical applications Biocompatibility Corrosion Mechanical properties Pores Eye glasses Biodegradability and bioabsorbable polymer High cell adhesion and less inflammation Wear resistance
2.8.3 Biomedical polymer composites Natural biomedical composites Synthetic biomedical composites
2.9 Applications of biocomposites
2.9.1 Tissue engineering
2.9.2 Orthopedic
2.9.3 Dental
2.9.4 External prosthetic and orthotics
2.9.5 Biocompatibility on skin
2.9.6 Healing of fracture and wound dressing
2.10 Fabrication techniques of biomedical composites
2.10.1 Hand layup molding
2.10.2 Open contact molding method
2.10.3 Liquid molding and injection molding
2.10.4 Vacuum resin transfer molding process
2.10.5 Compression molding
2.10.6 Tube rolling
2.10.7 Automated fiber/tape placement process
2.11 Conclusion
3. Biopolymer-based composites for drug delivery applications—a scientometric analysis
3.1 Introduction
3.2 Scientometric analysis
3.2.1 Coauthorship analysis
3.2.2 Cooccurrence analysis Chitosan Alginate Cellulose Hyaluronic acid
3.2.3 Analysis of the citations of the articles
4. Characteristicsand characterization techniques of bacterial cellulose for biomedical applications—a short treatise
4.1 Introduction
4.2 Biomedical applications of bacterial cellulose
4.2.1 Wound healing applications
4.2.2 Diagnosis of ovarian cancer
4.2.3 Shape memory material
4.2.4 Preventing deterioration of salmon muscle and slowing down the lipid oxidation
4.2.5 Lipase immobilization
4.2.6 Tissue engineering
4.2.7 Implantable devices in regenerative medicine
4.2.8 Drug delivery
4.2.9 Bone healing
4.2.10 Wound dressing
4.3 Conclusion
5. Engineering scaffolds for tissue engineering and regenerative medicine
5.1 Introduction
5.2 Scaffolds properties and characterization
5.3 Fabrication of scaffolds
5.3.1 Scaffold fabrication methods
5.3.2 Patient-specific scaffolds
Declaration of conflict of interest
6. Recenttrendsinpolymeric composites and blends for three-dimensional printing and bioprinting
6.1 Introduction
6.2 Need of synergistic approach in polymeric materials
6.3 Blends and composites of natural and synthetic polymers
6.3.1 Synthetic polymers based composites
6.3.2 Natural polymers based composites
6.4 3D printing techniques employed to print polymeric materials
6.4.1 Extrusion-based 3D printing Fused deposition modeling 3D plotting
6.4.2 Vat polymerization
6.4.3 Powder bed fusion Selective laser sintering Binder jetting or powder liquid 3D printing
6.4.4 Laser-assisted bioprinting
6.5 Application of value-added polymers
6.6 Current challenges and possible solutions
6.7 Conclusion
7. Polymers for additive manufacturing and 4D-printing for tissue regenerative applications
7.1 Introduction
7.2 Polymers for 4D printing
7.2.1 Hydrogels
7.2.2 Shape memory polymers
7.2.3 Elastomer actuators
7.2.4 Thermoresponsive polymers
7.3 Application of 4D printing technology
7.3.1 Engineered tissue constructs Soft tissue regenerative implants Hard tissue regenerative implants
7.3.2 Medical devices
7.3.3 Drug delivery implants
8. Bioprinting of hydrogels for tissue engineering and drug screening applications
8.1 Advancements in bioprinting technology
8.2 Bioinks
8.3 Hydrogel bioinks
8.4 Applications of hydrogel bioinks
8.4.1 Bone tissue engineering
8.4.2 Cartilage tissue engineering
8.4.3 Cardiac tissue engineering
8.4.4 Skin tissue engineering
8.4.5 Vascular tissue engineering
8.4.6 Neural tissue engineering
8.4.7 Drug screening
8.5 Challenges of bioprinted hydrogels in tissue engineering and drug screening
8.6 Conclusion and future perspectives
9. Smart polymers for biomedical applications
9.1 Introduction
9.2 Temperature-sensitive smart polymers
9.3 Applications of temperature-sensitive smart polymers
9.4 pH-sensitive smart polymers
9.4.1 Applications
9.5 Photosensitive polymers
9.5.1 Applications
9.6 Enzyme-responsive polymers
9.6.1 Applications
9.7 Conclusion
10. Chitosan-based nanoparticles for ocular drug delivery
10.1 Introduction
10.2 Anatomy and protection mechanism of eye
10.3 Properties of chitosan
10.4 Some recent applications of chitosan nanoparticles in ocular delivery
10.5 Conclusion
11. Appraisal of conducting polymers for potential bioelectronics
11.1 Introduction
11.2 Sensors and actuators used on conducting polymers
11.3 Energy storage from conducting polymer
11.4 Energy harvesting based on polymer
11.5 Organic light-emitting diodes
11.5 Organic light-emitting diodes
11.6 Electrochromic materials and devices
12. Shape-memory polymers
12.1 Introduction
12.2.1 Cross-linking
12.2 Various shape-memory polymers
12.2 Various shape-memory polymers
12.2.2 Thermal transitions
12.2.3 Categorization of shape-memory polymers
12.3 Mechanism of shape-memory polymers
12.4 Composites using shape-memory polymers
12.4.1 Functionalization of shape-memory polymers by silicate
12.4.2 Functionalization of shape-memory polymers by magnetic particles
12.4.3 Functionalization of shape-memory polymers by carbon fillers
12.4.4 Functionalization of shape-memory polymers by biocompatible mater
12.5 Limitations of shape-memory polymers
12.5.1 Recovery time and activation process
12.5.2 Recovery force and work capacity
12.6 Conclusion
13. Rapid prototyping
13.1 Introduction
13.2 Preprocessing, the process, and postprocessing in rapid prototyping
13.2.1 Preprocessing
13.2.2 The process
13.2.3 Postprocessing
13.3 Contemporary rapid prototyping systems
13.3.1 Available rapid prototyping systems Selective laser sintering Selective laser melting Laminated object manufacturing Fused deposition modeling (FDM) Stereolithography
13.4 Applications
13.5 Advancements in the rapid prototyping technology
13.5.1 Improvement of product quality
13.5.2 Improvement on versatility of rapid prototyping
13.5.3 Multifunctional fabrication process
13.5.4 Printable and embeddable functions Sensors Actuations Thermal management Energy storage Antennas and electromagnetic structures Propulsion
13.5.5 Fiber-reinforced polymer composites
13.5.6 Functionally graded materials using rapid prototyping
13.5.7 Comparison with traditional manufacturing
14. Self-assembled polymer nanocomposites in biomedical applications
14.1 Introduction
14.2 Methods of preparation of self-assembled polymer nanocomposites
14.2.1 Polymer grafting on/from the modified surface of nanoparticles
14.2.2 Layer-by layer assembly technique
14.3 Applications of the self-assembled polymer nanocomposites in biomedical science
14.3.1 Drug delivery
14.4 Future prospects and conclusion
15. Thermoresponsive polymers and polymeric composites
15.1 Introduction
15.1.1 Thermoresponsive polymers
15.1.2 Thermoresponsive polymeric composites
15.2 Mechanisms
15.2.1 Protein adsorption
15.2.2 Cells adhesion and attachments
15.2.3 Thermoresponsive behaviors Principle for thermoresponsive polymers showing UCST and LCST Type of thermoresponsive polymers Poly(N-alkyl-substituted acrylamide)s Poly(N-vinylcaprolactam) Poly(2-alkyl-2-oxazoline)s Poly(ether)s Poly(N,N-(dimethylamino)ethyl methacrylate) Poly(oligo(ethylene glycol) methyl ether methacrylate)s
15.3 Form of thermoresponsive polymers and polymeric composites
15.3.1 Hydrogels
15.3.2 Nanoparticles
15.3.3 Micelles
15.3.4 Films
15.3.5 Interpenetrating networks
15.3.6 Polymersomes
15.4 Applications of thermoresponsive polymers
15.4.1 Vascular applications
15.4.2 Gene delivery
15.4.3 Drug delivery
15.4.4 Wound healing Wound healing phases Hemostasis Inflammation Proliferation Tissue remodeling Application of thermoresponsive polymers in wound healing
15.5 Future perspectives
15.6 Conclusion
Further reading
16. Ceramic particle-dispersed polymer composites
16.1 Introduction
16.2 Matrices used in ceramic particle dispersed polymer composites
16.2.1 Biodegradable matrices Modification or recycling polymer matrices
16.2.2 Nonbiodegradable matrices Thermoplastics Thermosetting
16.3 Reinforcements used in ceramic particle reinforced composites
16.3.1 Reinforcement from natural resources
16.3.2 Reinforcements from synthetic resources
16.4 Fabrication of ceramic particulate dispersed composites
16.4.1 Methods of composite fabrication Methods for thermoplastics Low-pressure processing techniques Thermoplastic composites considering vacuum forming Autoclave forming of thermoplastic composites Diaphragm forming Bladder inflation molding Resin Transfer Moulding (RTM) Injection-compression technique High-pressure processing Preheating technology for stamp-forming processes Blank-holders and membrane forces Continuous compression molding Methods for thermosetting Open molding Closed molding
16.5 Curing of the composites
16.5.1 Room-temperature curing
16.5.2 High-temperature curing
16.6 Different types of ceramic particle dispersed composites
16.6.1 Particulate-reinforced composites
16.6.2 Hybrid composites
16.7 Characterization
16.7.1 Structural properties Scanning electron microscope (SEM) and field emission scanning electron microscope (FESEM) analysis
16.7.2 Charpy impact strength test
16.7.3 Atomic force microscopy Fourier transform infrared (FTIR) analysis Tensile testing Flexural testing Izod impact test Thermogravimetric analysis
16.8 Summary
17. Electrospinning for biomedical applications
17.1 Introduction
17.1.1 Theory of electrospinning
17.1.2 Principle of electrospinning
17.2 Parameters influencing fiber production
17.2.1 System parameters Applied voltage Flow rate Tip to collector distance Collector types
17.2.2 Solution parameters Concentration Surface tension Molecular weight Conductivity/surface charge density
17.2.3 Ambient parameters
17.3 Polymers for fabrication of electrospun fibers
17.3.1 Synthetic polymers Poly L-lactic-co-glycolic acid PLLA-polylactic acid Polycaprolactone Polyurethane
17.3.2 Natural polymers Gelatin Chitosan Silk
17.3.3 Composite and hybrid
17.4 Applications of electro-spun fibers in tissue engineering applications
17.4.1 Use of electro-spun polymers in neural tissue engineering Use of electro-spun fibers in cardiac tissue engineering
17.5 Conclusion
Further reading
18. Advances in biomedical polymers and composites: Drug delivery systems
18.1 Introduction
18.2 Synthesis of polymer composites
18.2.1 Hydrothermal method
18.2.2 In situ polymerization
18.2.3 Electrospinning method
18.2.4 Three-dimensional printing technology
18.3 Characterization and drug release properties
18.3.1 X-ray diffraction
18.3.2 Fourier transform infrared spectroscopy
18.3.3 Thermal analysis
18.3.4 Scanning electron microscopy
18.3.5 Determination of drug loading into composites
18.3.6 Estimation of drug release from composites
18.3.7 Mathematical treatment of drug release kinetics
18.3.8 Mechanisms for controlling drug release from composites
18.4 Applications in drug delivery
18.4.1 Tumor-targeted drug therapy
18.4.2 Ophthalmic drug delivery
18.4.3 Buccal drug delivery
18.4.4 Drug delivery for bone tissue regeneration
18.5 Conclusion and future perspectives
19. Natural gums of plant and microbial origin for tissue engineering applications
19.1 Introduction
19.2 Scientometric analysis
19.2 Scientometric analysis
19.3 Natural gums
19.3.1 Gellan gum Applications
19.3.2 Xanthan gum Applications
19.3.3 Guar gum Applications
19.4 Conclusion
20. Polymers and nanomaterials as gene delivery systems
20.1 Introduction
20.2 Types of gene delivery
20.2.1 Germline gene therapy
20.2.2 Somatic gene therapy Ex vivo delivery In situ delivery In vivo delivery
20.3 Methods and techniques used in gene delivery
20.3.1 Nanoparticle gene delivery systems Mesoporous silica nanoparticles
20.3.2 Liposome gene delivery systems
20.3.3 Microbubble gene delivery systems
20.3.4 Viral and nonviral gene delivery systems Viral gene delivery systems Nonviral gene delivery system
20.4 Polymers and bioceramics for gene delivery
20.4.1 Natural polymer chitosan
20.4.2 Synthetic polymers Thermoresponsive polymers Polyethylenimine
20.5 Applications of gene delivery
20.5.1 Cancer
20.5.2 Cardiovascular
20.5.3 Kidney
20.5.4 Bone
21. Essential oil-loaded biopolymeric films for wound healing applications
21.1 Introduction
21.2 Wound healing physiology
21.3 Essential oils
21.3.1 Mechanisms of promoting wound healing by essential oils
21.3.2 Methods of preparation of essential oil-loaded films
21.3.3 Essential oil-loaded biopolymeric films for wound healing applications
21.4 Conclusion
22. Biomedical antifouling polymer nanocomposites
22.1 Introduction
22.2 Mechanism of antifouling
22.2.1 Strategies of antifouling
22.2.2 Natural antifouling
22.3 Biomedical antifouling
22.3.1 Nanogel engineering
22.3.2 Zwitterionic nanomaterials
22.3.3 Superhydrophobic surfaces and wettability
22.4 Computational studies
22.4.1 Exploring the antifouling properties of polymers using computational methods
22.4.2 Effect of surface hydration on antifouling properties
22.4.3 Polyzwitterions
22.5 Conclusion
23. Application of antiviral activity of polymer
23.1 Introduction
23.2 Types of antiviral polymers
23.2.1 Polysaccharides
23.2.2 Antiviral peptide polymer
23.2.3 Nucleic acid polymers
23.2.4 Polymer-drug conjugates
23.2.5 Metal containing polymers
23.2.6 Dendrimers
23.3 Application of antiviral polymers
23.3.1 Drug delivery system
23.3.2 Polymers in protective application
23.3.3 Food packaging
23.4 Concluding remarks
24. Biosensor: fundamentals, biomolecular component, and applications
24.1 Introduction
24.2 Fundamentals of biosensor
24.2.1 Principle of biosensor
24.3 Classification of the biosensors
24.4 Characteristics of the biosensors
24.5 Biopolymers for the development of biosensors
24.5.1 Biopolymer composites
24.6 Biomolecular component of biosensor
24.7 Recent trends in biosensors
24.8 Recent applications of biosensors
24.9 Merits and limitation of biosensors
25. Polymeric materials in microbial cell encapsulation
25.1 Introduction
25.2 Encapsulation method
25.2.1 Nanoprecipitation
25.2.2 Emulsification
25.2.3 Coacervation
25.2.4 Capillary encapsulation method
25.2.5 Electrospinning
25.2.6 Layer-by-layer self-assembly method
25.2.7 Spray drying
25.3 Applications
25.3.1 Intestinal tract health
25.3.2 Bioavailability and nutrient synthesis
25.3.3 Probiotics’ antimicrobial potential
25.3.4 Cancer prevention
25.3.5 Tissue engineering
25.3.6 Methylene blue dye remediation from water
25.3.7 In Agriculture and the food processing
25.3.8 Drug delivery
25.4 Conclusion
25.5 Future considerations
26. Carbon nanotubes based composites for biomedical applications
26.1 Introduction
26.2 Carbon nanotube based composites for biomedical applications
26.2.1 Carbon nanotube nanocomposites for biosensors
26.2.2 Carbon nanotube nanocomposites for drug delivery
26.2.3 Carbon nanotube nanocomposites for cancer treatment
26.2.4 Carbon nanotube nanocomposites for tissue engineering
26.3 Toxicity of carbon nanotubes
26.4 Future prospective
26.5 Conclusion
Conflict of interest
27. Cryogels as smart polymers in biomedical applications
27.1 Introduction
27.2 What is cryogel?
27.3 Cryogel preparation method
27.4 The precursors in cryogel preparation
27.5 The cross-linking strategy in cryogel preparation
27.6 Characterization of cryogels
27.7 The biomedical applications of the cryogels
27.7.1 Cryogels in bioseparation process
27.7.2 Cryogels in wound dressing applications
27.7.3 Cryogels in tissue engineering applications Cryogels as bioreactors Cryogels in cell separations Cryogels as tissue scaffolds
27.7.4 Cryogels in drug release applications
27.8 Conclusion
28. Naturally derived ceramics-polymer composite for biomedical applications
28.1 Introduction
28.2 Preparation of biogenic-derived biocomposites
28.2.1 Materials
28.2.2 Various biocomposites from biowaste materials
28.2.3 Zinc-substituted hydroxyapatite/cellulose nanocrystals biocomposite
28.2.4 Hydroxyapatite reinforced with polyvinylpyrrolidone/aloe vera biocomposite
28.2.5 Hydroxyapatite/carboxymethyl cellulose/sodium alginate biocomposite
28.2.6 Characterization
• Bioactivity assessment
• Mechanical studies
• Antibacterial activity
• In vitro cell viability analysis
28.3 Results and discussion
28.3.1 Egg shell derived hydroxyapatite/cellulose nanocrystals biocomposite
• FTIR analysis
• XRD analysis
• SEM and EDX investigations
• Mechanical characterization
• Antibacterial activity
28.3.2 Crab shell extracted hydroxyapatite/poly (vinylpolypyrrolidone)/aloe vera biocomposite
• FTIR analysis
• XRD analysis
• SEM analysis
• Mechanical characterizations
• Contact angle measurements
• Antibacterial activity
• In vitro cytocompatibility analysis
28.3.3 Fish bone derived hydroxyapatite/biopolymer composite
• FTIR spectroscopic analysis
• X-ray diffraction investigation
• Microstructural evaluation
• In vitro bioactivity assessment
• Microhardness analysis
• Antibacterial analysis
28.4 Conclusion
29. Molecularly imprinted polymers (MIPs) for biomedical applications
29.1 Introduction
29.2 Molecular imprinting technology
29.2.1 Key parameters for the preparation of molecularly imprinted polymers
29.2.2 Approaches for the preparation of molecularly imprinted polymers
29.3 Applications of molecularly imprinted polymers in biomedical science
29.3.1 Drug delivery
29.3.2 Bio-imaging and cancer therapy
29.3.3 Sensing and separation processes
29.4 Conclusions and future perspectives
30. Natural biopolymer scaffolds for bacteriophage delivery in the medical field
30.1 Introduction
30.2 Phage therapy
30.2.1 Regulatory approval of phage therapy
30.2.2 Phage application in medicine
30.3 Bacteriophage encapsulation
30.3.1 Encapsulation of phages in natural polymers
30.3.2 Phage encapsulation for wound healing applications
30.3.3 Phage encapsulation to prevent and manage gastrointestinal diseases
30.4 Conclusions and future perspectives

Citation preview

Advances in Biomedical Polymers and Composites Materials and Applications

Advances in Biomedical Polymers and Composites Materials and Applications Edited by

Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha, India

Sarika Verma Materials for Radiation Shielding and Cement Free Concrete Division, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Bhopal, India

Pallab Datta Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India

Ananya Barui Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, India

S. A. R. Hashmi Integrated Approach for Design and Product Development Devision, CSIR-Advanced Materials and Processes Research Institute (AMPRI), Bhopal, India

Avanish Kumar Srivastava CSIR-Advanced Materials and Processes Research Institute, Bhopal, India

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: 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: 978-0-323-88524-9 For Information on all Elsevier publications visit our website at

Publisher: Matthew Deans Acquisitions Editor: Edward Payne Editorial Project Manager: Sara Greco Production Project Manager: Surya Narayanan Jayachandran Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Contents List of contributors .................................................................................................xxi

CHAPTER 1 Introduction to biomedical polymer and composites .................................................................... 1 1.1 1.2 1.3


1.5 1.6

Soham Chowdhury, Adhish Singh and Bidyut Pal Introduction ....................................................................................1 Classification of polymers and composites ...................................2 Fabrication techniques polymer composites..................................3 1.3.1 Electrospinning ................................................................... 4 1.3.2 Melt extrusion ..................................................................... 4 1.3.3 Solution mixing................................................................... 4 1.3.4 Latex technology................................................................. 6 Polymers and their composites for biomedical applications.........7 1.4.1 Natural polymers and their composites .............................. 7 1.4.2 Synthetic polymers and their composites......................... 13 1.4.3 Gas-permeable polymeric membranes ............................. 18 1.4.4 Other polymeric composites ............................................. 19 Challenges and future trends........................................................22 Conclusion ....................................................................................23 References.................................................................................... 24

CHAPTER 2 Foundation of composites........................................... 31 2.1 2.2 2.3

2.4 2.5

Umesh Kumar Dwivedi and Neelam Kumari Introduction ..................................................................................31 Classification of composites ........................................................32 History of composites ..................................................................33 2.3.1 Fiberglass in 20th century................................................. 34 2.3.2 Composite material in our daily life ................................ 35 Why composites? .........................................................................35 Advantages of composites ...........................................................35 2.5.1 Design flexibility ............................................................ 35 2.5.2 Light weight .................................................................... 36 2.5.3 High strength................................................................... 36 2.5.4 Strength related to weight............................................... 36 2.5.5 Corrosion resistance........................................................ 36 2.5.6 High-impact strength ...................................................... 36 2.5.7 Consolidation of many parts........................................... 37 2.5.8 Dimensional stability ...................................................... 37





2.7 2.8




2.5.9 Nonconductive ................................................................ 37 2.5.10 Nonmagnetic ................................................................... 37 2.5.11 Radar transparent ............................................................ 37 2.5.12 Low thermal conductivity............................................... 38 2.5.13 Durable ............................................................................ 38 Applications of composites..........................................................38 2.6.1 Aerospace/aircrafts............................................................ 38 2.6.2 Appliances......................................................................... 38 2.6.3 Automobile and transportation ......................................... 38 2.6.4 Infrastructure ..................................................................... 38 2.6.5 Environmental ................................................................... 39 2.6.6 Applications of electricity................................................. 39 Limitation of composites .............................................................39 Biocomposites and classification .................................................40 2.8.1 Biomedical composites ..................................................... 40 2.8.2 Basic requirements and parameters for biomedical applications ....................................................................... 40 2.8.3 Biomedical polymer composites....................................... 42 Applications of biocomposites.....................................................43 2.9.1 Tissue engineering ............................................................ 45 2.9.2 Orthopedic......................................................................... 46 2.9.3 Dental ................................................................................ 47 2.9.4 External prosthetic and orthotics ...................................... 49 2.9.5 Biocompatibility on skin................................................... 51 2.9.6 Healing of fracture and wound dressing .......................... 51 Fabrication techniques of biomedical composites.......................54 2.10.1 Hand layup molding........................................................ 54 2.10.2 Open contact molding method........................................ 54 2.10.3 Liquid molding and injection molding........................... 55 2.10.4 Vacuum resin transfer molding process ......................... 55 2.10.5 Compression molding ..................................................... 56 2.10.6 Tube rolling..................................................................... 57 2.10.7 Automated fiber/tape placement process........................ 57 Conclusion ....................................................................................58 References.................................................................................... 58

CHAPTER 3 Biopolymer-based composites for drug delivery applications—a scientometric analysis .................... 61 Kunal Pal, Deepti Bharti, Preetam Sarkar and Doman Kim 3.1 Introduction ..................................................................................61


3.2 Scientometric analysis..................................................................62 3.2.1 Coauthorship analysis ....................................................... 64 3.2.2 Cooccurrence analysis ...................................................... 66 3.2.3 Analysis of the citations of the articles ............................ 75 3.3 Conclusion ....................................................................................80 References.................................................................................... 80

CHAPTER 4 Characteristics and characterization techniques of bacterial cellulose for biomedical applications—a short treatise ................................... 83 Kumar Anupam, Richa Aggrawal, Jitender Dhiman, Priti Shivhare Lal, Thallada Bhaskar and Dharm Dutt 4.1 Introduction ..................................................................................83 4.2 Biomedical applications of bacterial cellulose ............................84 4.2.1 Wound healing applications ........................................... 86 4.2.2 Diagnosis of ovarian cancer ........................................... 93 4.2.3 Shape memory material .................................................. 93 4.2.4 Preventing deterioration of salmon muscle and slowing down the lipid oxidation ................................... 94 4.2.5 Lipase immobilization .................................................... 94 4.2.6 Tissue engineering .......................................................... 94 4.2.7 Implantable devices in regenerative medicine ............... 98 4.2.8 Drug delivery .................................................................. 99 4.2.9 Bone healing ................................................................. 100 4.2.10 Wound dressing............................................................. 100 4.3 Conclusion ..................................................................................105 References.................................................................................. 106

CHAPTER 5 Engineering scaffolds for tissue engineering and regenerative medicine.............................................. 109 5.1 5.2 5.3


Ibrahim Fatih Cengiz, Rui L. Reis and Joaquim Miguel Oliveira Introduction ................................................................................109 Scaffolds properties and characterization ..................................110 Fabrication of scaffolds..............................................................114 5.3.1 Scaffold fabrication methods .......................................... 114 5.3.2 Patient-specific scaffolds ................................................ 117 Conclusion ..................................................................................120 Acknowledgments ..................................................................... 120 Declaration of conflict of interest ............................................. 121 References.................................................................................. 121




CHAPTER 6 Recent trends in polymeric composites and blends for three-dimensional printing and bioprinting ....... 131 6.1 6.2 6.3


6.5 6.6 6.7

Sriya Yeleswarapu, K.N. Vijayasankar, Shibu Chameettachal and Falguni Pati Introduction ................................................................................131 Need of synergistic approach in polymeric materials ...............132 Blends and composites of natural and synthetic polymers .......133 6.3.1 Synthetic polymers based composites .......................... 134 6.3.2 Natural polymers based composites ............................. 135 3D printing techniques employed to print polymeric materials .....................................................................................138 6.4.1 Extrusion-based 3D printing ........................................... 139 6.4.2 Vat polymerization.......................................................... 140 6.4.3 Powder bed fusion........................................................... 141 6.4.4 Laser-assisted bioprinting ............................................... 143 Application of value-added polymers........................................143 Current challenges and possible solutions.................................145 Conclusion ..................................................................................147 References.................................................................................. 147

CHAPTER 7 Polymers for additive manufacturing and 4D-printing for tissue regenerative applications .... 159 7.1 7.2



Bhuvaneshwaran Subramanian, Pratik Das, Shreya Biswas, Arpita Roy and Piyali Basak Introduction ................................................................................159 Polymers for 4D printing ...........................................................161 7.2.1 Hydrogels ........................................................................ 162 7.2.2 Shape memory polymers ................................................ 163 7.2.3 Elastomer actuators ......................................................... 164 7.2.4 Thermoresponsive polymers ........................................... 165 Application of 4D printing technology......................................166 7.3.1 Engineered tissue constructs........................................... 166 7.3.2 Medical devices .............................................................. 169 7.3.3 Drug delivery implants ................................................... 170 Conclusion ..................................................................................178 Reference ................................................................................... 178

CHAPTER 8 Bioprinting of hydrogels for tissue engineering and drug screening applications ............................. 183 Ece O¨zmen, O¨zu¨m Yıldırım and Ahu Arslan-Yıldız 8.1 Advancements in bioprinting technology ..................................183


8.2 Bioinks........................................................................................186 8.3 Hydrogel bioinks ........................................................................188 8.4 Applications of hydrogel bioinks...............................................193 8.4.1 Bone tissue engineering .................................................. 198 8.4.2 Cartilage tissue engineering............................................ 198 8.4.3 Cardiac tissue engineering .............................................. 203 8.4.4 Skin tissue engineering ................................................... 203 8.4.5 Vascular tissue engineering ............................................ 204 8.4.6 Neural tissue engineering................................................ 205 8.4.7 Drug screening ................................................................ 206 8.5 Challenges of bioprinted hydrogels in tissue engineering and drug screening .....................................................................209 8.6 Conclusion and future perspectives ...........................................210 References.................................................................................. 211

CHAPTER 9 Smart polymers for biomedical applications........... 223 9.1 9.2 9.3 9.4 9.5 9.6 9.7

Deepti Bharti, Indranil Banerjee, Preetam Sarkar, Doman Kim and Kunal Pal Introduction ................................................................................223 Temperature-sensitive smart polymers ......................................225 Applications of temperature-sensitive smart polymers .............226 pH-sensitive smart polymers......................................................228 9.4.1 Applications .................................................................... 230 Photosensitive polymers.............................................................232 9.5.1 Applications .................................................................... 234 Enzyme-responsive polymers ....................................................237 9.6.1 Applications .................................................................... 238 Conclusion ..................................................................................240 References.................................................................................. 241

CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery...................................................................... 247 10.1 10.2 10.3 10.4 10.5

Kunal Pal, Bikash K. Pradhan, Doman Kim and Maciej Jarze˛bski Introduction ................................................................................247 Anatomy and protection mechanism of eye ..............................248 Properties of chitosan.................................................................250 Some recent applications of chitosan nanoparticles in ocular delivery............................................................................254 Conclusion ..................................................................................257 References.................................................................................. 258




CHAPTER 11 Appraisal of conducting polymers for potential bioelectronics ........................................................... 265 11.1 11.2 11.3 11.4 11.5 11.6 11.7

Rimita Dey and Pallab Datta Introduction ................................................................................265 Sensors and actuators used on conducting polymers ................267 Energy storage from conducting polymer .................................272 Energy harvesting based on polymer.........................................279 Organic light-emitting diodes ....................................................283 Electrochromic materials and devices .......................................285 Conclusions ................................................................................289 References.................................................................................. 289

CHAPTER 12 Shape-memory polymers .......................................... 299 Deepshikha Rathore 12.1 Introduction ................................................................................299 12.2 Various shape-memory polymers ..............................................300 12.2.1 Cross-linking ................................................................. 300 12.2.2 Thermal transitions ....................................................... 301 12.2.3 Categorization of shape-memory polymers.................. 302 12.3 Mechanism of shape-memory polymers....................................302 12.4 Composites using shape-memory polymers ..............................303 12.4.1 Functionalization of shape-memory polymers by silicate ........................................................................... 304 12.4.2 Functionalization of shape-memory polymers by magnetic particles .................................................... 304 12.4.3 Functionalization of shape-memory polymers by carbon fillers ................................................................. 306 12.4.4 Functionalization of shape-memory polymers by biocompatible materials........................................... 307 12.5 Limitations of shape-memory polymers ....................................308 12.5.1 Recovery time and activation process .......................... 308 12.5.2 Recovery force and work capacity ............................... 310 12.6 Conclusion ..................................................................................312 References.................................................................................. 312

CHAPTER 13 Rapid prototyping...................................................... 315 Umesh K. Dwivedi, Shashank Mishra and Vishal Parashar 13.1 Introduction ................................................................................315 13.2 Preprocessing, the process, and postprocessing in rapid prototyping .................................................................................319 13.2.1 Preprocessing ................................................................ 319


13.3 13.4 13.5


13.2.2 The process ................................................................... 320 13.2.3 Postprocessing ............................................................... 320 Contemporary rapid prototyping systems..................................321 13.3.1 Available rapid prototyping systems ............................ 323 Applications................................................................................329 Advancements in the rapid prototyping technology..................334 13.5.1 Improvement of product quality ................................... 335 13.5.2 Improvement on versatility of rapid prototyping ......... 335 13.5.3 Multifunctional fabrication process.............................. 336 13.5.4 Printable and embeddable functions............................. 336 13.5.5 Fiber-reinforced polymer composites........................... 337 13.5.6 Functionally graded materials using rapid prototyping .................................................................... 338 13.5.7 Comparison with traditional manufacturing................. 338 Conclusion ..................................................................................339 References.................................................................................. 339

CHAPTER 14 Self-assembled polymer nanocomposites in biomedical applications ........................................... 343 14.1 14.2



Anurag Dutta, Manash Jyoti Baruah, Satyabrat Gogoi and Jayanta Kumar Sarmah Introduction ................................................................................343 Methods of preparation of self-assembled polymer nanocomposites ..........................................................................345 14.2.1 Polymer grafting on/from the modified surface of nanoparticles ................................................................. 346 14.2.2 Layer-by layer assembly technique .............................. 347 Applications of the self-assembled polymer nanocomposites in biomedical science ......................................350 14.3.1 Drug delivery ................................................................ 350 Future prospects and conclusion................................................356 References.................................................................................. 357

CHAPTER 15 Thermoresponsive polymers and polymeric composites ................................................................ 363 Mh Busra Fauzi, Samantha Lo, Maheswary Thambirajoo, Zawani Mazlan, Izzat Zulkiflee, Syafira Masri, Isma Liza Mohd Isa and Sabarul Afian Mokhtar 15.1 Introduction ................................................................................363 15.1.1 Thermoresponsive polymers ......................................... 363 15.1.2 Thermoresponsive polymeric composites .................... 364 15.2 Mechanisms ................................................................................366






15.5 15.6

15.2.1 Protein adsorption ......................................................... 366 15.2.2 Cells adhesion and attachments.................................... 369 15.2.3 Thermoresponsive behaviors ........................................ 371 Form of thermoresponsive polymers and polymeric composites ..................................................................................374 15.3.1 Hydrogels ...................................................................... 375 15.3.2 Nanoparticles................................................................. 376 15.3.3 Micelles ......................................................................... 377 15.3.4 Films.............................................................................. 378 15.3.5 Interpenetrating networks ............................................. 379 15.3.6 Polymersomes ............................................................... 380 Applications of thermoresponsive polymers .............................381 15.4.1 Vascular applications .................................................... 381 15.4.2 Gene delivery ................................................................ 383 15.4.3 Drug delivery ................................................................ 384 15.4.4 Wound healing .............................................................. 385 Future perspectives.....................................................................388 Conclusion ..................................................................................390 References.................................................................................. 390 Further reading .......................................................................... 397

CHAPTER 16 Ceramic particle dispersed polymer composites ................................................................ 399 16.1 16.2


16.4 16.5


Bhabatosh Biswas, Gurudas Mandal, Apurba Das, Abhijit Majumdar and Arijit Sinha Introduction ................................................................................399 Matrices used in ceramic particle dispersed polymer composites ..................................................................................401 16.2.1 Biodegradable matrices................................................. 401 16.2.2 Nonbiodegradable matrices .......................................... 404 Reinforcements used in ceramic particle reinforced composites ..................................................................................405 16.3.1 Reinforcement from natural resources ......................... 405 16.3.2 Reinforcements from synthetic resources .................... 405 Fabrication of ceramic particulate dispersed composites........406 16.4.1 Methods of composite fabrication ................................ 406 Curing of the composites ...........................................................411 16.5.1 Room-temperature curing ............................................. 411 16.5.2 High-temperature curing ............................................... 411 Different types of ceramic particle dispersed composites ..................................................................................412


16.6.1 Particulate-reinforced composites................................. 412 16.6.2 Hybrid composites ........................................................ 415 16.7 Characterization..........................................................................416 16.7.1 Structural properties...................................................... 416 16.7.2 Charpy impact strength test .......................................... 418 16.7.3 Atomic force microscopy.............................................. 419 16.8 Summary.....................................................................................424 References.................................................................................. 424

CHAPTER 17 Electrospinning for biomedical applications........... 433 17.1





Srividya Hanuman, Steffi Zimran, Manasa Nune and Goutam Thakur Introduction ................................................................................433 17.1.1 Theory of electrospinning............................................. 433 17.1.2 Principle of electrospinning .......................................... 434 Parameters influencing fiber production ...................................436 17.2.1 System parameters ........................................................ 437 17.2.2 Solution parameters ...................................................... 438 17.2.3 Ambient parameters ...................................................... 440 Polymers for fabrication of electrospun fibers ..........................440 17.3.1 Synthetic polymers........................................................ 440 17.3.2 Natural polymers........................................................... 444 17.3.3 Composite and hybrid ................................................... 449 Applications of electro-spun fibers in tissue engineering applications.................................................................................450 17.4.1 Use of electro-spun polymers in neural tissue engineering .................................................................... 450 Conclusion ..................................................................................455 References.................................................................................. 455 Further reading .......................................................................... 463

CHAPTER 18 Advances in biomedical polymers and composites: Drug delivery systems ..................................................... 465 Aalok Basu and Amit Kumar Nayak 18.1 Introduction ................................................................................465 18.2 Synthesis of polymer composites ..............................................466 18.2.1 Hydrothermal method ................................................... 466 18.2.2 In situ polymerization ................................................... 467 18.2.3 Electrospinning method ................................................ 468 18.2.4 Three-dimensional printing technology........................ 469




18.3 Characterization and drug release properties ............................470 18.3.1 X-ray diffraction ........................................................... 470 18.3.2 Fourier transform infrared spectroscopy ...................... 471 18.3.3 Thermal analysis ........................................................... 472 18.3.4 Scanning electron microscopy ...................................... 472 18.3.5 Determination of drug loading into composites........... 473 18.3.6 Estimation of drug release from composites................ 473 18.3.7 Mathematical treatment of drug release kinetics ......... 474 18.3.8 Mechanisms for controlling drug release from composites..................................................................... 476 18.4 Applications in drug delivery ....................................................477 18.4.1 Tumor-targeted drug therapy ........................................ 478 18.4.2 Ophthalmic drug delivery ............................................. 479 18.4.3 Buccal drug delivery..................................................... 479 18.4.4 Drug delivery for bone tissue regeneration .................. 483 18.5 Conclusion and future perspectives ...........................................484 References.................................................................................. 485

CHAPTER 19 Natural gums of plant and microbial origin for tissue engineering applications ......................... 497 Kunal Pal, Deepti Bharti, Goutam Thakur and Doman Kim 19.1 Introduction ................................................................................497 19.2 Scientometric analysis................................................................499 19.3 Natural gums ..............................................................................500 19.3.1 Gellan gum.................................................................... 501 19.3.2 Xanthan gum ................................................................. 504 19.3.3 Guar gum....................................................................... 506 19.4 Conclusion ..................................................................................508 References.................................................................................. 509

CHAPTER 20 Polymers and nanomaterials as gene delivery systems...................................................................... 513 Sundara Ganeasan M, Amulya Vijay, M. Kaviya, Anandan Balakrishnan and T.M. Sridhar 20.1 Introduction ................................................................................513 20.2 Types of gene delivery...............................................................514 20.2.1 Germline gene therapy.................................................. 515 20.2.2 Somatic gene therapy.................................................... 516 20.3 Methods and techniques used in gene delivery.........................517 20.3.1 Nanoparticle gene delivery systems ............................. 517 20.3.2 Liposome gene delivery systems.................................. 520


20.3.3 Microbubble gene delivery systems ............................. 521 20.3.4 Viral and nonviral gene delivery systems .................... 523 20.4 Polymers and bioceramics for gene delivery ............................526 20.4.1 Natural polymer chitosan.............................................. 527 20.4.2 Synthetic polymers........................................................ 527 20.5 Applications of gene delivery ....................................................531 20.5.1 Cancer............................................................................ 531 20.5.2 Cardiovascular............................................................... 532 20.5.3 Kidney ........................................................................... 533 20.5.4 Bone .............................................................................. 534 20.6 Conclusion ..................................................................................535 Acknowledgment ....................................................................... 536 References.................................................................................. 536

CHAPTER 21 Essential oil-loaded biopolymeric films for wound healing applications ..................................... 541 21.1 21.2 21.3


Kunal Pal, Preetam Sarkar, Goutam Thakur and Doman Kim Introduction ................................................................................541 Wound healing physiology ........................................................542 Essential oils...............................................................................546 21.3.1 Mechanisms of promoting wound healing by essential oils .................................................................. 550 21.3.2 Methods of preparation of essential oil-loaded films............................................................................... 552 21.3.3 Essential oil-loaded biopolymeric films for wound healing applications ...................................................... 554 Conclusion ..................................................................................557 References.................................................................................. 558

CHAPTER 22 Biomedical antifouling polymer nanocomposites...... 563 Javad B.M. Parambath, Mahreen Arooj and Ahmed A. Mohamed 22.1 Introduction ................................................................................563 22.2 Mechanism of antifouling ..........................................................567 22.2.1 Strategies of antifouling................................................ 567 22.2.2 Natural antifouling ........................................................ 568 22.3 Biomedical antifouling...............................................................569 22.3.1 Nanogel engineering ..................................................... 569 22.3.2 Zwitterionic nanomaterials ........................................... 573 22.3.3 Superhydrophobic surfaces and wettability.................. 576




22.4 Computational studies ................................................................577 22.4.1 Exploring the antifouling properties of polymers using computational methods ....................................... 577 22.4.2 Effect of surface hydration on antifouling properties....................................................................... 578 22.4.3 Polyzwitterions.............................................................. 579 22.5 Conclusion ..................................................................................581 Acknowledgments ..................................................................... 582 References.................................................................................. 582

CHAPTER 23 Application of antiviral activity of polymer ............. 591 Shradha Sharma, Howa Begam and Ananya Barui 23.1 Introduction ................................................................................591 23.2 Types of antiviral polymers .......................................................592 23.2.1 Polysaccharides ............................................................. 592 23.2.2 Antiviral peptide polymer............................................. 595 23.2.3 Nucleic acid polymers .................................................. 597 23.2.4 Polymer-drug conjugates .............................................. 597 23.2.5 Metal containing polymers ........................................... 599 23.2.6 Dendrimers .................................................................... 599 23.3 Application of antiviral polymers ..............................................601 23.3.1 Drug delivery system .................................................... 601 23.3.2 Polymers in protective application ............................... 603 23.3.3 Food packaging ............................................................. 607 23.4 Concluding remarks ...................................................................609 References.................................................................................. 610

CHAPTER 24 Biosensor: fundamentals, biomolecular component, and applications ................................... 617

24.1 24.2 24.3 24.4 24.5 24.6 24.7

Manoj Kumar Tripathi, C. Nickhil, Adinath Kate, Rahul M. Srivastva, Debabandya Mohapatra, Rajpal S. Jadam, Ajay Yadav and Bharat Modhera Introduction ................................................................................617 Fundamentals of biosensor.........................................................618 24.2.1 Principle of biosensor ................................................... 618 Classification of the biosensors .................................................618 Characteristics of the biosensors................................................621 Biopolymers for the development of biosensors .......................622 24.5.1 Biopolymer composites ................................................ 623 Biomolecular component of biosensor ......................................625 Recent trends in biosensors........................................................626


24.8 Recent applications of biosensors..............................................627 24.9 Merits and limitation of biosensors ...........................................630 References.................................................................................. 630

CHAPTER 25 Polymeric materials in microbial cell encapsulation............................................................ 635 25.1 25.2


25.4 25.5

Memoona Akhtar, Muhammad Farrukh Sarfraz, Samra Fatima and Muhammad Atiq Ur Rehman Introduction ................................................................................635 Encapsulation method ................................................................637 25.2.1 Nanoprecipitation .......................................................... 638 25.2.2 Emulsification ............................................................... 638 25.2.3 Coacervation ................................................................. 639 25.2.4 Capillary encapsulation method ................................... 639 25.2.5 Electrospinning ............................................................. 640 25.2.6 Layer-by-layer self-assembly method .......................... 642 25.2.7 Spray drying .................................................................. 643 Applications................................................................................644 25.3.1 Intestinal tract health..................................................... 644 25.3.2 Bioavailability and nutrient synthesis .......................... 645 25.3.3 Probiotics’ antimicrobial potential ............................... 646 25.3.4 Cancer prevention ......................................................... 646 25.3.5 Tissue engineering ........................................................ 647 25.3.6 Methylene blue dye remediation from water ............... 647 25.3.7 In Agriculture and the food processing ........................ 647 25.3.8 Drug delivery ................................................................ 648 Conclusion ..................................................................................649 Future considerations .................................................................649 References.................................................................................. 650

CHAPTER 26 Carbon nanotubes based composites for biomedical applications ........................................... 657 Sarika Verma, Ramesh Rawat, Vaishnavi Hada, Ram Krishna Shrivastava, Kunal Pal, Sai S. Sagiri, Medha Mili, S. A. R. Hashmi and A.K. Srivastava 26.1 Introduction ................................................................................657 26.2 Carbon nanotube based composites for biomedical applications.................................................................................659 26.2.1 Carbon nanotube nanocomposites for biosensors ........ 660 26.2.2 Carbon nanotube nanocomposites for drug delivery.......................................................................... 661




26.2.3 Carbon nanotube nanocomposites for cancer treatment........................................................................ 663 26.2.4 Carbon nanotube nanocomposites for tissue engineering .................................................................... 663 26.3 Toxicity of carbon nanotubes ....................................................665 26.4 Future prospective ......................................................................665 26.5 Conclusion ..................................................................................666 Acknowledgment ....................................................................... 667 Conflict of interest..................................................................... 667 References.................................................................................. 667

CHAPTER 27 Cryogels as smart polymers in biomedical applications............................................................... 675 27.1 27.2 27.3 27.4 27.5 27.6 27.7


O¨zlem Bic¸en U¨nlu¨er, Ru¨stem Kec¸ili, Rıdvan Say and Arzu Erso¨z Introduction ................................................................................675 What is cryogel?.........................................................................676 Cryogel preparation method.......................................................677 The precursors in cryogel preparation .......................................681 The cross-linking strategy in cryogel preparation.....................682 Characterization of cryogels ......................................................683 The biomedical applications of the cryogels.............................686 27.7.1 Cryogels in bioseparation process ................................ 687 27.7.2 Cryogels in wound dressing applications ..................... 690 27.7.3 Cryogels in tissue engineering applications ................. 692 27.7.4 Cryogels in drug release applications........................... 700 Conclusion ..................................................................................704 References.................................................................................. 704

CHAPTER 28 Naturally derived ceramics polymer composite for biomedical applications ..................................... 711 E. Shinyjoy, S. Ramya, P. Saravanakumar, P. Manoravi, L. Kavitha and D. Gopi 28.1 Introduction ................................................................................711 28.2 Preparation of biogenic-derived biocomposites ........................716 28.2.1 Materials........................................................................ 716 28.2.2 Various biocomposites from biowaste materials ......... 716 28.2.3 Zinc-substituted hydroxyapatite/cellulose nanocrystals biocomposite ............................................ 717 28.2.4 Hydroxyapatite reinforced with polyvinylpyrrolidone/aloe vera biocomposite .............. 718


28.2.5 Hydroxyapatite/carboxymethyl cellulose/sodium alginate biocomposite ................................................... 718 28.2.6 Characterization ............................................................ 718 28.3 Results and discussion................................................................721 28.3.1 Egg shell derived hydroxyapatite/cellulose nanocrystals biocomposite ............................................ 722 28.3.2 Crab shell extracted hydroxyapatite/poly (vinylpolypyrrolidone)/aloe vera biocomposite ........... 725 28.3.3 Fish bone derived hydroxyapatite/biopolymer composite ...................................................................... 730 28.4 Conclusion ..................................................................................739 Acknowledgments ..................................................................... 739 References.................................................................................. 740

CHAPTER 29 Molecularly imprinted polymers (MIPs) for biomedical applications ........................................... 745 29.1 29.2



Ru¨stem Kec¸ili, O¨zlem Bic¸en U¨nlu¨er, Arzu Erso¨z and Rıdvan Say Introduction ................................................................................745 Molecular imprinting technology ..............................................746 29.2.1 Key parameters for the preparation of molecularly imprinted polymers ....................................................... 746 29.2.2 Approaches for the preparation of molecularly imprinted polymers ....................................................... 749 Applications of molecularly imprinted polymers in biomedical science .....................................................................751 29.3.1 Drug delivery ................................................................ 751 29.3.2 Bio-imaging and cancer therapy................................... 755 29.3.3 Sensing and separation processes ................................. 758 Conclusions and future perspectives..........................................762 References.................................................................................. 763

CHAPTER 30 Natural biopolymer scaffolds for bacteriophage delivery in the medical field .................................... 769 Ana Mafalda Pinto, Marisol Dias, Lorenzo M. Pastrana, Miguel A. Cerqueira and Sanna Sillankorva 30.1 Introduction ................................................................................769 30.2 Phage therapy .............................................................................769 30.2.1 Regulatory approval of phage therapy ......................... 773 30.2.2 Phage application in medicine...................................... 773




30.3 Bacteriophage encapsulation......................................................776 30.3.1 Encapsulation of phages in natural polymers............... 777 30.3.2 Phage encapsulation for wound healing applications ................................................................... 780 30.3.3 Phage encapsulation to prevent and manage gastrointestinal diseases ................................................ 782 30.4 Conclusions and future perspectives..........................................784 Funding ...................................................................................... 786 References.................................................................................. 786 Index ......................................................................................................................795

List of contributors Richa Aggrawal Department of Chemical Engineering, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Haryana, India Memoona Akhtar Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Kumar Anupam Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India; Chemical Recovery and Biorefinery Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India Mahreen Arooj Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Ahu Arslan-Yıldız Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey Anandan Balakrishnan Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, Chennai, Tamil Nadu, India Indranil Banerjee Department of Bioscience & Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India Manash Jyoti Baruah Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India Ananya Barui Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Piyali Basak School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Aalok Basu Department of Pharmaceutics, Dr. BC Roy College of Pharmacy and Allied Health Sciences, Durgapur, West Bengal, India Howa Begam Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Deepti Bharti Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India



List of contributors

Thallada Bhaskar Material Resource Efficiency Division, CSIR-Indian Institute of Petroleum, Dehradun, Uttarakhand, India; Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India O¨zlem Bic¸en U¨nlu¨er Faculty of Sciences, Chemistry Department, Eskis¸ehir Technical University, Eskis¸ehir, Turkey Bhabatosh Biswas Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, Delhi, India Shreya Biswas School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Ibrahim Fatih Cengiz 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal Miguel A. Cerqueira INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Shibu Chameettachal Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Soham Chowdhury Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Apurba Das Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Pratik Das School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India Pallab Datta Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India Rimita Dey Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India Jitender Dhiman Biotechnology Division, Central Pulp, and Paper Research Institute, Saharanpur, Uttar Pradesh, India

List of contributors

Marisol Dias INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Dharm Dutt Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India Anurag Dutta Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India; Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India Umesh K. Dwivedi Amity School of Applied Sciences, Amity University Jaipur, Jaipur, Rajasthan, India Umesh Kumar Dwivedi Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India Arzu Erso¨z Chemistry Department, Faculty of Science, Eskis¸ehir Technical University, Eskis¸ehir, Turkey; Bionkit Co Ltd., Eskis¸ehir, Turkey Samra Fatima Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Mh Busra Fauzi Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Sundara Ganeasan M Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India Satyabrat Gogoi Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India D. Gopi Department of Chemistry, Periyar University, Salem, Tamil Nadu, India Vaishnavi Hada Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India Srividya Hanuman Manipal Institute of Regenerative Medicine, Bangalore, Karnataka, India; Manipal Academy of Higher Education, Manipal, Karnataka, India S. A. R. Hashmi Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India



List of contributors

Rajpal S. Jadam ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Maciej Jarze˛bski Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznan´ University of Life Sciences, Poznan´, Poland Adinath Kate ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India L. Kavitha Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India M. Kaviya Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India Ru¨stem Kec¸ili Department of Medical Services and Techniques, Yunus Emre Vocational School of Health Services, Anadolu University, Eskis¸ehir, Turkey Doman Kim Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea Neelam Kumari Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India Priti Shivhare Lal Physical Chemistry, Pulping and Bleaching Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India Samantha Lo Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Abhijit Majumdar Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, Delhi, India Gurudas Mandal Department of Metallurgical Engineering, Kazi Nazrul University, Asansol, West Bengal, India P. Manoravi Materials Chemistry and Metal Fuel Cycle Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamil Nadu, India Syafira Masri Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

List of contributors

Zawani Mazlan Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Medha Mili Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Shashank Mishra Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India Bharat Modhera Department of Biotechnology, MANIT, Bhopal, Madhya Pradesh, India Ahmed A. Mohamed Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Debabandya Mohapatra ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Isma Liza Mohd Isa Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Sabarul Afian Mokhtar Department of Orthopaedics and Traumatology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Amit Kumar Nayak Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Odisha, India C. Nickhil Department of Food Engineering & Technology, Tezpur University, Tezpur, Assam, India Manasa Nune Manipal Institute of Regenerative Medicine, Bangalore, Karnataka, India; Manipal Academy of Higher Education, Manipal, Karnataka, India Joaquim Miguel Oliveira 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal Ece O¨zmen Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey



List of contributors

Bidyut Pal Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India Javad B.M. Parambath Department of Chemistry, College of Sciences, University of Sharjah, Sharjah, United Arab Emirates Vishal Parashar Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India Lorenzo M. Pastrana INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Falguni Pati Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Ana Mafalda Pinto Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, Portugal; INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Bikash K. Pradhan Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India S. Ramya Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Deepshikha Rathore Amity School of Applied Sciences, Amity University Rajasthan, Jaipur, Rajasthan, India Ramesh Rawat Department of Chemistry, Institute for Excellence in Higher Education, Bhopal, Madhya Pradesh, India Muhammad Atiq Ur Rehman Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Rui L. Reis 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal; ICVS/3B’s–PT Government Associate Laboratory, Guimara˜es, Portugal

List of contributors

Arpita Roy Polymer Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology (ISM), Dhanbad, Jharkhand, India Sai S. Sagiri Department of Food Science, Agricultural Research Organization, AgroNanotechnology and Advanced Materials Research Center, the Volcani Institute, Rishon Lezion, Israel P. Saravanakumar Department of Chemistry, Periyar University, Salem, Tamil Nadu, India Muhammad Farrukh Sarfraz Department of Materials Science and Engineering, Institute of Space Technology Islamabad, Islamabad, Pakistan Preetam Sarkar Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India Jayanta Kumar Sarmah Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India Rıdvan Say Department of Chemistry, Faculty of Sciences, Anadolu University, Eskis¸ehir, Turkey; Bionkit Co Ltd., Eskis¸ehir, Turkey Shradha Sharma Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India E. Shinyjoy Department of Physics, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Ram Krishna Shrivastava Department of Chemistry, Institute for Excellence in Higher Education, Bhopal, Madhya Pradesh, India Sanna Sillankorva INL—International Iberian Nanotechnology Laboratory, Braga, Portugal Adhish Singh Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India Arijit Sinha Department of Metallurgical Engineering, Kazi Nazrul University, Asansol, West Bengal, India T.M. Sridhar Department of Analytical Chemistry, University of Madras, Chennai, Tamil Nadu, India



List of contributors

A.K. Srivastava Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Rahul M. Srivastva Department of Biotechnology, MANIT, Bhopal, Madhya Pradesh, India Bhuvaneshwaran Subramanian School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India Goutam Thakur Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Maheswary Thambirajoo Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Manoj Kumar Tripathi ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India O¨zlem Bic¸en U¨nlu¨er Chemistry Department, Faculty of Science, Eskis¸ehir Technical University, Eskis¸ehir, Turkey Sarika Verma Council of Scientific and Industrial Research-Advanced Materials and Processes Research Institute, Bhopal, Madhya Pradesh, India; Academy of Council Scientific and Industrial Research (AcSIR), Advanced Materials and Processes Research Institute (AMPRI), Bhopal, Madhya Pradesh, India Amulya Vijay Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, Chennai, Tamil Nadu, India K.N. Vijayasankar Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India Ajay Yadav ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India Sriya Yeleswarapu Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India O¨zu¨m Yıldırım Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey

List of contributors

Steffi Zimran Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Izzat Zulkiflee Center for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia



Introduction to biomedical polymer and composites


Soham Chowdhury, Adhish Singh and Bidyut Pal Department of Mechanical Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, West Bengal, India

1.1 Introduction Synthetic or natural materials that can be used in conjunction with or as a substitute for living tissues of the human body are called biomaterials (Ulery, Nair, & Laurencin, 2011). Advancements in synthetic materials and fabrication techniques, aided further by cutting-edge sterilization and surgical procedure, have established the clinical use of biomaterials in the current medical practice (Ramakrishna, Mayer, Wintermantel, & Leong, 2001). Biomaterials are extensively used to replace and/or regenerate the function of degenerate or damaged tissues and organs, thereby assisting in healing and improving the functioning and the overall quality of life of the patient (Salernitano & Migliaresi, 2003). Since the biological tolerance of the same material is host-dependent, the term “biocompatibility” was coined to characterize the suitability of materials in a clinical setting. The term “biocompatibility” suggests that when implanted in living tissue, the implant material can produce desirable biological and chemical reactions (Wintermantel, Mayer, Ruffieux, Bruinink, & Eckert, 1999). This necessitates both surface and structure compatibility. A material is surface-compatible when the chemistry, biology, and surface morphology of the implant surface are suitable to the host tissue. Structural compatibility is characterized by an optimal interaction at the implant/tissue interface when the implant material adapts to the mechanical behavior of the surrounding host tissue. Desirable interactions between the biomaterial and the host tissue are achieved when both the surface compatibility and the structural compatibility are met. Biomaterials that are being used in the form of implants (bone plates, joint replacements, sutures, bone graft, vascular grafts, dental implants, etc.) and medical devices (artificial hearts, pacemakers, biosensors, etc.) are anticipated to perform satisfactorily in the aggressive environment of the body (Camilo et al., 2017). Scaffolds developed not only have to have sufficient mechanical strength to bear the structural loads but should also possess an internal porous network that will facilitate tissue ingrowth, cell proliferation, and vascularization (Mayer, Karamuk, Akaike, & Wintermantel, 2000). The material used in the scaffold must actively interact with the surrounding tissue to prevent immune rejection. Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 1 Introduction to biomedical polymer and composites

Needless to say, the physiological environment of a patient also depends upon their conditions and activities. Recently, there has been an increased interest in the development of partially or completely resorbable biomaterials for clinical applications to replace the bio-stable materials that are currently being used. Significant research has been carried out to evaluate biodegradable polymeric materials’ efficacy for various biomedical applications (Karamuk, Mayer, Wintermantel, & Akaike, 1999). Degradable polymeric biomaterials were found to be useful for developing temporary prostheses and degradable porous scaffolds for replacing or regenerating damaged tissues. They can also be used in drug carriers for controlled release of the drug. Polymeric materials are being used extensively for applications in tissue engineering due to their availability in a wide variety of compositions, forms (fiber, gel, solid, fabrics, and films), properties and ease of fabrication into complex shapes and structures (Dhand et al., 2016). Tissues are generally categorized into hard and soft tissues. As the name suggests, the hard tissues (bone, tooth) are stiffer than soft tissues (skin, ligament, cartilage, blood vessels). Thus metals and ceramics are generally used for hard tissue applications, and polymers are used for the latter. However, as the load carried by a material is directly related to its stiffness, metals and ceramics pose several complications in orthopedic applications such as stress-shielding, implant loosening, periprosthetic fracture, etc., due to the stiffness mismatch with the neighboring bone tissue. It has been reported that matching the stiffness of the implant material to the neighboring host tissue can significantly reduce the stress-shielding as well as promote favorable bone remodeling around the implant (Apostu, Lucaciu, Berce, Lucaciu, & Cosma, 2017). Although polymers seem like an interesting choice due to their low modulus of elasticity, they cannot be used as they are not capable of sustaining the mechanical loads observed at the implantation site on account of their low strength (Park, Oh, & Lee, 2018). To overcome the shortcomings of using homogeneous materials in isolation, composite biomaterials are being developed. Human tissues are composite materials exhibiting anisotropic properties, which depend on the orientation and structure of the components (Ramakrishna et al., 2001). Polymer composite materials, that is, fiber-reinforced polymers, are characterized by low elastic modulus along with high strength, making them suitable for orthopedic applications. The functionality and performance of a composite implant can be made to optimally adapt to the mechanical and physiological properties of the neighboring host tissue by controlling the composition and orientation of the reinforcement phase. This chapter presents a brief overview of the characteristics, fabrication techniques, and biomedical applications of polymeric materials and polymer composites.

1.2 Classification of polymers and composites Polymeric biomaterials can be broadly grouped into natural polymers and synthetic polymers (Choudhary, Saraswat, & Venkatraman, 2019). The natural polymers can be further grouped into proteins, polysaccharides, and polynucleotides. Among them, proteins and polysaccharides are most commonly used for applications in tissue engineering.

1.3 Fabrication techniques polymer composites

FIGURE 1.1 Classification of composite materials based on the nature and dispersion of the reinforcement phase. Adopted from Wang, M., & Zhao, Q. (2019). Biomedical composites. In: Encyclopedia of biomedical engineering (pp. 34 52). Elsevier, with permission from Elsevier.

Quite a few composites have been evaluated for applications in the medical industry. A broad classification of composite materials is shown in Fig. 1.1. According to the type of the matrix phase, composites can be classified into metal composites, ceramic composites, and polymer composites (Wang & Zhao, 2019). Generally, the characteristics of the composite are determined by the type of matrix. For example, polymer matrix composites are usually ductile in nature. Alternatively, based on the type of reinforcement, composites can be categorized into fiber-reinforced, particle-reinforced, and structural composites. Particlereinforced composites are further categorized as large-particle and dispersionstrengthened materials, depending on the size of the particulate reinforcement. The fiber-reinforced composites can be further subcategorized based on the length of the fiber (long fiber and short fiber). The long fibers are dispersed in a continuous and aligned manner within the composite, whereas in the case of the short fibers, the dispersion can either be regular or randomly oriented. Fiber-reinforced composites are favored due to the enhancement in strength and controllability of the mechanical properties by adjusting the composition, length, orientation, and concentration of the fibers. The structural composites are composed of multiple homogeneous composite layers and can be further grouped into laminates and sandwich panels. Laminates are constructed by a sequential stacking and cementing of multiple anisotropic layers. Due to their unique fabrication, laminates possess excellent in-plane stiffness. Sandwich panels are fabricated by sandwiching a low-density layer with two stiff face sheets. Sandwich panels are generally lightweight and have excellent resistance to bending.

1.3 Fabrication techniques polymer composites Polymer composites are fabricated using various processes. The processes are designed to fabricate polymer composites suitable for various applications, including biomedical applications. Selected techniques are discussed in the following sections.



CHAPTER 1 Introduction to biomedical polymer and composites

1.3.1 Electrospinning The electrospinning technique utilizes high-strength electric fields to process ultrafine fibers up to 2 nm in diameter. The fabrication of nanofibers with a large surface area is performed using this technique in comparison with generally used spinning techniques. A coaxial electrospinning system is illustrated in Fig. 1.2. The basic components of an electrospinning system are spinneret, high-voltage power supply, and grounded collector (Ponnamma, Sadasivuni, Cabibihan, & AlMaadeed, 2017; Zagho & Elzatahry, 2016). Electrospinning can be divided into two types, namely, vertical and horizontal sets (Ponnamma et al., 2017; Zagho & Elzatahry, 2016). When a high voltage is applied to the polymeric solution, a charged liquid jet is formed from the Taylor cone tip. As the solvent evaporates, the liquid jet dries, resulting in the formation of fibers.

1.3.2 Melt extrusion For the production of polymeric composites, the most frequently utilized technique is melting extrusion. Polymer and fillers are mixed together with the help of a twin-screw extruder, which is used for a specific period at a specific temperature (Ponnamma et al., 2017). Compression molding is used to give shape to the polymer composites after the extrusion to get the final product. The amount of filler present determines the mechanical and thermal properties of the polymer composite produced by this technique. Nanocomposites were prepared by Majeed, Al Ali AlMaadeed, and Zagho (2018) having 3 wt.% nanotubes and 97 wt.% low-density polyethylene (LDPE) mixed at 180 C for 7 min. The preparation of titania nanotubes from commercially available TiO2 powder is illustrated in Fig. 1.3. LDPE galleries and the nanotubes are found to be incompatible; therefore maleic anhydride polyethylene can be utilized as compatible material. The mixture was then compression molded for 5 min at 180 C and 200 MPa, to produce thin films (Majeed et al., 2018).

1.3.3 Solution mixing The solution mixing technique fabricates polymer composites by dissolving polymers into a certain solvent at a fixed temperature (Ponnamma et al., 2017). This is followed by fillers being homogeneously distributed in the polymer solution. The mixture is constantly stirred at a fixed temperature. Then a mold is used to dry the resulting mixture at a specified temperature (Zepp et al., 2020). Al-Marri, Masoud, Nassar, Zagho, and Khader (2015) synthesized poly(vinyl alcohol) (PVA)/Cloisite 20A polymeric composites by utilizing PVA polymer solution and suspending Cloisite 20A filler at 70 C for 30 min. A square aluminum mold was used to dry the mixture at 25 C. The PVA polymer solution was prepared by mixing PVA pellets in chloroform and constant stirring at 60 C for 2 h (Al-Marri et al., 2015).

1.3 Fabrication techniques polymer composites

FIGURE 1.2 (A) Schematic illustration of a coaxial electrospinning system, (B) overview of the fabrication process of alginate hydrogel composites reinforced with polycaprolactone nanofibers, and (C) optical images of the fabricated composites with 0.0, 0.023, 0.085, and 0.122 volume fraction of nanofibers, respectively (Jang, Lee, Seol, Jeong, & Cho, 2013). Adopted from Jang, J., Lee, J., Seol, Y.-J., Jeong, Y. H., & Cho, D.-W. (2013). Improving mechanical properties of alginate hydrogel by reinforcement with ethanol treated polycaprolactone nanofibers. Composites Part B: Engineering, 45, 1216 1221, with permission from Elsevier.



CHAPTER 1 Introduction to biomedical polymer and composites

FIGURE 1.3 (A) Scanning electron micrographs of commercially available TiO2 powder; (B) scanning electron micrograph, (C) transmission electron micrograph, and (D) X-ray diffraction analysis (XRD) diffractograms of hydrothermally prepared titania nanotubes. Adopted from Majeed, K., Al Ali AlMaadeed, M., & Zagho, M. M. (2018). Comparison of the effect of carbon, halloysite and titania nanotubes on the mechanical and thermal properties of LDPE based nanocomposite films. Chinese Journal of Chemical Engineering, 26, 428 435, with permission from Elsevier.

1.3.4 Latex technology Latex technology is used to fabricate conductive polymer composites. This is achieved by incorporating conductive fillers into the polymer network. Process upscaling and easy processing make this technique advantageous. The polymer network shows homogeneously distributed fillers (Grossiord, Hermant, & Tkalya, 2012). Also, the highly viscous polymer network can take the nanofillers when using the technique. The three steps involved in this process are as follows: disperse nanofillers into a colloidal mixture, the addition of polymer latex, and drying

1.4 Polymers and their composites for biomedical applications

of colloidal mixture (Grossiord et al., 2012). Carbon nanotubes (Rode, Sharma, & Mishra, 2018) and graphene polymer composites (Pei, Ai, & Qu, 2015), which have various biomedical applications, can be fabricated using this technique.

1.4 Polymers and their composites for biomedical applications 1.4.1 Natural polymers and their composites Collagen Collagen (Col), a naturally occurring protein, is the major constituent of both the soft tissue (skin, cartilage, blood vessels) and the hard tissue (teeth, bone). It provides strength and structurally supports the tissues (Lee, Singla, & Lee, 2001). There are 29 different types of Col, out of which Ⅰ, Ⅱ, Ⅲ, Ⅴ, and ⅩⅠ types are widely studied for purposes of tissue repairing. Since Type Ⅰ Col does not elicit an allergic response, it has found multiple applications in tissue engineering (Parenteau-Bareil, Gauvin, & Berthod, 2010). The biocompatibility, flexibility, and biodegradability make Col a suitable polymer for biomedical applications. Studies have found that Col sponges used as scaffolds assist in the adhesion and growth of cells and tissues (Freyman, Yannas, & Gibson, 2001; O’Brien, Harley, Yannas, & Gibson, 2005). Moreover, it promotes osteoblast proliferation and induces osteoblast differentiation, thereby enhancing bone formation (Seol et al., 2004). A study was conducted to evaluate the performance of Col gel infused with mesenchymal cells in the repair of osteochondral defects. At the implant site, the formation of hyaline cartilage and bone, having mechanical stability significantly lower than the surrounding host tissue, was observed (Wakitani et al., 1994). Studies indicate that the biological properties of Col depend on the orientation of the Col fibers. A study was reported comparing the activity of random Col fiber scaffolds and aligned Col fiber scaffolds fabricated using the electrospinning technique. It was observed that rabbit conjunctival fibroblast cells proliferated faster on the aligned fibrous scaffolds. The rapid degradation rate of Col protein results in poor mechanical properties (Zhong et al., 2006). To overcome this shortcoming, Col composites are made using natural (glycosaminoglycans) and synthetic polymers (polyglycerol methacrylate), characterized by good mechanical strength and osteoconductivity, for promoting the repair of bone tissue (Daamen, 2003; Woerly, Marchand, & Lavalle´e, 1991). Silk Silk fibroin is a natural polymeric protein processed from silkworms (Bombyx mori) and insects. The biocompatibility characteristics associated with silk, such as high strength, biodegradability, flexibility, and permeability to water and oxygen make it a promising biomaterial for tissue engineering. The application of


CHAPTER 1 Introduction to biomedical polymer and composites


silk is hindered majorly due to sericin protein, a common contaminant that elicits adverse reactions at the site of application (Puppi, Chiellini, Piras, & Chiellini, 2010). Studies indicated that silk has the potential to enhance the proliferation and differentiation of osteoblastic cells and simulate chondrogenesis and osteogenesis from mesenchymal cells extracted from bone marrow (Meinel et al., 2004; Vepari & Kaplan, 2007). The efficacy of silk sericin in the preparation of wound dressing gauze is illustrated in Fig. 1.4. A study investigated the biological activity of silk fibroin hydrogel postapplication for treating cancellous defects in rabbits. The results indicated an increased healing rate of the defective bone and enhanced quality of the newly formed bone tissue when silk fibroin hydrogel was present, compared to the control material, commercial synthetic poly(D,L-lactide glycolide) copolymer (Fini et al., 2005). Semi-IPN nanocomposite hydrogel H2N H HO





















FIGURE 1.4 (A) Sericin/poly(NIPAm/LMSH) nanocomposite hydrogel dressings were applied at wound sites in rats, (B) wound healing after treating with gauze, HSP00, and HSP20 nanocomposite hydrogel dressings, and (C) histological evaluation done on the skin at the 13th day by hematoxylin and eosin (H&E) staining, after treatment with gauze, HSP00, and HSP20 nanocomposite hydrogel dressings (Yang et al., 2017). Adopted from Yang, C., Xue, R., Zhang, Q., Yang, S., Liu, P., Chen, L., . . . Wei, Y. (2017). Nanoclay crosslinked semi-IPN silk sericin/poly(NIPAm/LMSH) nanocomposite hydrogel: An outstanding antibacterial wound dressing. Materials Science and Engineering: C, 81, 303 313, with permission from Elsevier.

1.4 Polymers and their composites for biomedical applications Hyaluronic acid Hyaluronic acid (HA), also known as hyaluronan, is a biodegradable polysaccharide that naturally occurs in the extracellular matrix of connective tissues. It assists in structural support and plays an important role in maintaining water balance and lubrication between articulating cartilage surfaces. HA is mainly extracted from vitreous humor, synovial fluid, and umbilical cord (Malafaya, Silva, & Reis, 2007). The viscoelasticity, swelling capability, and poor immunogenic response of HA make it a potential biomaterial for applications in the encapsulation of cells and drug delivery systems (Kang et al., 2009; Wieland, Houchin-Ray, & Shea, 2007). Moreover, the extensive availability, biodegradability, biocompatibility, and amenability to chemical modification have made HA a potential polymer for tissue engineering (Allison & Grande-Allen, 2006). Studies indicated that the polyanionic and hydrophilic surface of HA does not favor attachment of anionic cell surfaces, thereby impeding cell growth and tissue remodeling (Shu, Liu, Palumbo, & Prestwich, 2003). To overcome this limitation, Shu et al. (2003) coated the surface of HA with extracellular matrix proteins, resulting in a significant improvement in cellular attachment and tissue formation. Further expansion of its applications has been achieved by modification of its chemical characteristics by photo linking and covalent crosslinking (Allison & Grande-Allen, 2006). In a recent study, the in vitro performance of HA-based Hyaft-ll biodegradable polymer infused with human vascular endothelial cells was evaluated. Formation of the subendothelial matrix was observed within 24 h (Fig. 1.5). The study indicates Hyaft-ll-based biopolymers can be used for making scaffolds that facilitate endothelialization in vascular grafts (Turner, Kielty, Walker, & Canfield, 2004). Chitosan Naturally occurring Chitosan is a biodegradable and biocompatible polysaccharide. It has wide applications in pharmaceutics, cosmetics, and the food industry (Perinelli et al., 2018). Chitosan is produced by partial deacetylation chitin, obtained mainly from the cuticles of various crustaceans through chemical hydrolysis (Chandy & Sharma, 1990). The extensive availability, antibacterial activity, hydrophilicity, and nonimmunogenic properties indicate why Chitosan is preferred for tissue engineering applications (Nair & Laurencin, 2007). Researchers have recently developed an antibacterial and biocompatible derivative of chitosan, 1,3-diethyl-2-thiobarbituric acid (CS-DETBA). CS-DETBA showed enhanced inhibition to the growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus bacteria and was observed to have no cytotoxic effects on the human gastric adenocarcinoma (AGS) cells (Rizwan et al., 2017). A study was carried out to determine the proliferation of mesenchymal stem cells produced from bone marrow in a chitosan/tripolyphosphate scaffold (Fig. 1.6). From the results, it was concluded that the developed scaffold has the potential to be used in bone regenerative medicine (Xu et al., 2018). Under in vivo and in vitro



CHAPTER 1 Introduction to biomedical polymer and composites

FIGURE 1.5 Confocal micrographs illustrating attachment of rat BM-MSCs on chitosan scaffolds after 24 h of culture in (A) 1%(w/w) TPP, (B) 3 M NaCl 1 1% (w/w) TPP, (C) 6 M NaCl 1 1% (w/w) TPP, (D) 3 M NaCl 1 0.25% (w/w) TPP, (E) 3 M NaCl 1 2%(w/w) TPP, and (F) 3 M NaCl 1 6% (w/w) TPP. Adopted from Turner, N. J., Kielty, C. M., Walker, M. G., & Canfield, A. E. (2004). A novel hyaluronan-based biomaterial (Hyaff-11®) as a scaffold for endothelial cells in tissue engineered vascular grafts. Biomaterials, 25, 5955 5964, with permission from Elsevier.

conditions, chitosan stimulates bone formation due to its osteoconductive ability. However, its poor mechanical stability restricts the maintenance of precise shape, thereby reducing its application. Cellulose Cellulose is a naturally occurring polysaccharide found in the cell walls of plants. Cellulose is involved in the provision of structural support. It also occurs in other microorganisms like fungi, algae, and bacteria (Puppi et al., 2010). The hydrophilicity and biocompatible characteristics of cellulose make it a potential biomaterial for application in tissue engineering and drug delivery (Klemm, Heublein, Fink, & Bohn, 2005). Cellulose hydrogel membranes are prepared by casting cellulose/1-butyl-3methylimidazolium chloride into assembly molds, followed by coagulation in

1.4 Polymers and their composites for biomedical applications

FIGURE 1.6 Confocal micrographs illustrating attachment of rat bone marrow mesenchymal stem cells on Chitosan scaffolds after 24 h of culture in (A) 1%(w/w) tripolyphosphate (TPP), (B) 3 M NaCl 1 1% (w/w) TPP, (C) 6 M NaCl 1 1% (w/w) TPP, (D) 3 M NaCl 1 0.25% (w/w) TPP, (E) 3 M NaCl 1 2%(w/w) TPP, and (F) 3 M NaCl 1 6% (w/w) TPP. Red represents the actin network with rhodamine-phalloidin and nuclei, and chitosan shown in blue and green, respectively. Adopted from Xu, Y., Han, J., Chai, Y., Yuan, S., Lin, H., & Zhang, X. (2018). Development of porous chitosan/tripolyphosphate scaffolds with tunable uncross-linking primary amine content for bone tissue engineering. Materials Science and Engineering: C, 85, 182 190, with permission from Elsevier.

water. Depending on the techniques adopted, porous cellulose hydrogels possessing suitable morphological features and mechanical properties can be prepared for applications in the delivery of pharmaceutical agents as a drug carrier, contact lenses, or wound healing material (Peng, Wang, Xu, & Dai, 2017). In a study, it was observed that cellulose enhances the proliferation and growth of human chondrocytes, as illustrated in Fig. 1.7, and thus can be utilized in cartilage tissue engineering (Svensson et al., 2005). In a recent study, a cellulose-based composite reinforced with silver and chitosan nanoparticles was fabricated. The composite was found to possess an excellent antibacterial inhibition to the growth of E. coli and S. aureus, and an increased proliferation rate of fibroblastic cells was observed within an incubation period of 3 days (Haider et al., 2018). The findings indicate this composite can be used for preparing scaffolds for wound dressing. An effective polysaccharide capsule has been developed for orally administering a hydrophobics drug (Ibuprofen) by physical crosslinking of hydroxyethyl



CHAPTER 1 Introduction to biomedical polymer and composites

FIGURE 1.7 Scanning electron microscopic (SEM) micrographs of bovine chondrocyte cell attachment on unmodified bacterial cellulose (BC) and modified BC (BC-P1, BC-P2, BC-S) surfaces. Adopted from Svensson, A., Nicklasson, E., Harrah, T., Panilaitis, B., Kaplan, D. L., Brittberg, M., & Gatenholm, P. (2005). Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials, 26, 419 431, Adopted with permission from Elsevier.

cellulose (HEC) and carboxymethyl cellulose (CMC). It was observed that the drug was completely released from HEC into the intestinal fluid within 8 h. However, upon mixing of HEC and CMC resulted in a prolonged (24 h) and sustained release of the drug from the carrier (Chen, Wang, & Yan, 2017). The performance of gelatin-based chitosan and hydroxyethyl cellulose was evaluated for applications in tissue engineering. A reduction in stiffness, enhanced flexibility, and mechanical strength comparable to soft tissues were observed for the gelatin poly(ethylene glycol) (PEG)/hydroxyethyl cellulose (G/PEG/HEC) hydrogel. A biological evaluation of the hydrogel was reported on rat myoblasts and human fibroblasts cell lines. The results showed good cell adhesion and enhanced proliferation, indicating its potential for biomedical applications (Dey et al., 2018).

1.4 Polymers and their composites for biomedical applications

1.4.2 Synthetic polymers and their composites Polycaprolactone Polycaprolactone (PCL) is a polymer made up of semipolar ester groups and nonpolar methylene groups. It finds its use in tissue engineering as it is biocompatible and has high elasticity. The Food and Drug Administration (FDA) gave approval to PCL for its use in various biomedical applications, which include drug delivery systems, tissue repair as sutures and scaffolds, etc. (Woodruff & Hutmacher, 2010). The slower degradation rate of PCL makes it a better material for implants and a drug carrier (Cipitria, Skelton, Dargaville, Dalton, & Hutmacher, 2011). When used as a bulk material, PCL shows slow adhesion and cell proliferation. In order to increase the bioactivity of PCL, its polymeric composites are being attempted. Apart from that, surface functionalization is also utilized for the same. A blend of PCL, chitosan-1,3-diethyl-2-thiobarbituric acid-PCL (CS-DETBAPCL), is prepared for its use in tissue engineering. The cytotoxicity response on AGS cells of this blend is negligible, and the growth of bacterial strains (S. aureus, E. coli, P. aeruginosa) was significantly hampered (Xu et al., 2018). Recently, with the help of the electrospinning method, a PCL/chitosan/magnesium oxide nanofiber was manufactured. Mechanical stability of PCL/MgO (25 MPa) is better than that of PCL/ chitosan (3 MPa). A cellular study suggests the noncytotoxic nature of the composites due to the attachment of 3T3 cells on its surface. Also, this study endorses PCL as a suitable polymer for bone regeneration and wound healing (Rijal et al., 2018). With or without bioactive ceramics, PCL can be utilized either way in hard tissue regeneration as a scaffold material. Interaction of bone marrow mesenchymal stem cells (BMSCs) with pure PCL indicates an insignificant influence on its functioning by the degradation by-products of PCL (Sukanya & Mohanan, 2018). PCL/forsterite scaffold was fabricated, which, as compared to pure PCL, showed improvement in bioactivity and cytotoxicity of scaffolds that were used for bone regeneration. The content of forsterite in the composites affects the cellular behavior of the composites (Diba, Tapia, Boccaccini, & Strobel, 2012). To repair the orbital fractures of the white rabbit, a thin membrane patch of PCL/β-tricalcium phosphate was three-dimensional (3-D) printed. It was observed that a 40% reduction of fracture volume took place after 2 months. Also, within 4 months, the mesh implant showed growth of new bone. This study pointed out that the use of the 3-D printed membrane patch for filling defective spaces in the bone. This could be an encouraging methodology as an inflammatory response was prevented at the site of application (Ho Han et al., 2018). Poly(L-lactic acid) Poly(L-lactic acid) (PLLA) can be manufactured from the polymerization of L-lactide or derived from natural sources. It is a degradable polymer. The use of PLLA as scaffolds for tissue regeneration, drug delivery carrier, and pin in fixing implants in bone and sutures has been heavily investigated. FDA approved Sculptra for commercial use in facial atrophy treatment in which PLLA is used as



CHAPTER 1 Introduction to biomedical polymer and composites

an injectable material (Stratton, Shelke, Hoshino, Rudraiah, & Kumbar, 2016). However, PLLA showed the inflammatory response at the site of application as it undergoes rapid degeneration due to its high crystalline nature (Lasprilla, Martinez, Lunelli, Jardini, & Filho, 2012). When fabricated with the other polymers as a composite, this issue can be resolved. Cui et al. (2013) prepared novel PLLA/Rg3 scaffolds that help reduce inflammation and also studied the response of composite to skin regeneration. The proliferation of fibroblast cells was hindered due to uniform surface morphology and interconnected pores of the scaffolds. This points out the ability of fabricated composites to restore damaged skin due to severe burns. With the help of the electrospinning technique, a defect-free fiber of PLLA/polyglycerol sebacate (PGS) was manufactured (Cui et al., 2013). Superhydrophilicity was achieved due to the use of PGS in fibers. The elastic modulus was observed to drop sharply from 35.9 to 7.4 MPa when there was an increase in 25% concentration of PGS in the fibers. Also, the stretching ability was improved by twofolds. A study found PLLA/PGS to be a promising biomaterial for nerve regeneration as it showed adhesion and proliferation of A59 nerve cells, as illustrated in Fig. 1.8

FIGURE 1.8 Scanning electron micrographs illustrating the distribution of A59 cells on the membrane surfaces for 1-day cell culture on (A) poly(L-lactic acid) (PLLA) membrane, and (B) PLLA: polyglycerol sebacate (PGS) (25 wt.% PGS) membrane and 5-day cell culture on (C) PLLA membrane, and (D) PLLA:PGS (25 wt.% PGS) membrane. Adopted from Yan, Y., Sencadas, V., Jin, T., Huang, X., Chen, J., Wei, D., & Jiang, Z. (2017). Tailoring the wettability and mechanical properties of electrospun poly(l-lactic acid)-poly(glycerol sebacate) core-shell membranes for biomedical applications. Journal of Colloid and Interface Science, 508, 87 94, with permission from Elsevier.

1.4 Polymers and their composites for biomedical applications

(Yan et al., 2017). A study was conducted by dual coating the Mg alloy with hydroxyapatite and PLLA in order to improve its biomedical application (Diez, Kang, Kim, Kim, & Song, 2015). The dual-coated alloys enhanced the biological response and mechanical stability in comparison to single-coated or noncoated samples. Poly(methyl methacrylate) Poly(methyl methacrylate) (PMMA) is a synthetic polymer with superior mechanical properties and self-hardening ability, which helps to fix an artificial joint to the bone (Lee & Rhee, 2009). Due to its inert nature, PMMA is a suitable material to provide immediate structural support to metallic implants in bone. However, in between an implant and bone, the PMMA is found to be a weak link (Renterı´a-Zamarro´n, Corte´s-Herna´ndez, Bretado-Arago´n, & Ortega-Lara, 2009). Repeated interfacial movements lead to osteolysis and loosening of the implants (Goodman, 2005). With the help of bioactive ceramic as filler in between the polymer matrix, the osteoconductivity and mechanical stability of the composite can be improved (Shinzato et al., 2000). Hydroxyapatite reinforced with PMMA provides improved anchorage of human osteoblast cells, increased activity of alkaline phosphatase, and cell proliferation (Dalby, Di Silvio, Harper, & Bonfield, 1999). In a study, Renterı´aZamarro´n et al. (2009) found good apatite forming ability and compressive strength in PMMA containing 39% wollastonite. Another study showed the use of PMMA/SiO2 CaO nanocomposites in dental composites as a filler material and bone cement (Lee & Rhee, 2009). Poly(lactic-co-glycolic) acid Poly(lactic-co-glycolic) acid (PLGA) is a biodegradable polyester. It is formed by combining PLLA and poly(glycolic acid) (PGA). PLGA has been the center of attention of researchers due to its biocompatibility and modifiable nature of its surface properties which encourage its interaction with biological materials and make it a right fit for tissue engineering applications (Gentile, Chiono, Carmagnola, & Hatton, 2014). FDA approved Osteofoam as a PLGA scaffold for hard tissue regeneration (Shen, Hu, Bei, & Wang, 2008). To treat craniosynostosis, a uniform mix of PLGA and polyisoprene was studied. The scaffolds were made with porous structures with pores of a size that promotes the growth of C2C12 cell lines and forms an extracellular matrix. Marques, dos Santos, O’Brien, Cartmell, and Gough (2016) proposed to use these scaffolds for soft tissue engineering as their strength was similar to that of natural soft tissue. For tendon regeneration, PLGA/silk scaffolds exhibited good mechanical stability and also the tendency to simulate mesenchymal progenitor cells to undergo adhesion and differentiation (Fig. 1.9) (Sahoo, Toh, & Goh, 2010). For its use in hard tissue engineering, PLGA/silk composites supported by hydroxyapatite were manufactured (Sheikh et al., 2015). It was observed that over the surface of



CHAPTER 1 Introduction to biomedical polymer and composites

FIGURE 1.9 (A) Unseeded and bone marrow mesenchymal stem cell seeded scaffold specimens at mechanical testing, (B) load displacement curves for the biohybrid scaffolds obtained after mechanical testing, (C) loads at which the representative scaffolds fail, and (D) stiffness obtained for the scaffolds. Adopted from Sahoo, S., Toh, S. L., & Goh, J. C. H. (2010). A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials, 31, 2990 2998, with permission from Elsevier.

microspheres of chitosan/PLGA, there was a proliferation of MC3T3-E1 (Jiang, Abdel-Fattah, & Laurencin, 2006). PLGA finds limited use in drug delivery applications due to the acidic nature of its by-products of degradation. Research is being done to resolve this issue by changing the concentration of PGA. To slow the degradation rate, the ratio of PGA to that of PLLA was increased, producing less acidic by-products (Houchin & Topp, 2008). Polyvinylidene fluoride Polyvinylidene fluoride (PVDF) is one of the most common fluorinated polymers. It has a semicrystalline structure and is a nonreactive polymer. Fluoride polymers have exceptional biocompatibility, thermal stability, chemical resistance, and stimulus-response that make them biomaterials with many biomedical applications (Cardoso, Correia, Ribeiro, Fernandes, & Lanceros-Me´ndez, 2018). For hard tissue regeneration, the ability of piezoelectric PVDF was examined by implanting PVDF films in Wistar rats. After a period of 28 days, the implanted

1.4 Polymers and their composites for biomedical applications

films led to remarkable bone regeneration without any inflammatory response (Ribeiro et al., 2017). PVDF finds its use in a variety of pharmaceutical applications and hygienic products. Due to these uses of PVDF, it is exposed to microorganisms that can form biofilms. To examine its antibacterial properties against P. aeruginosa, different PVDF composites were prepared with several nanofillers. Nanofillers used in the composites were graphene nanoplatelets (GNPs), zinc oxide nanorods (ZnO-NRs), and ZnO-NR-decorated GNPs (ZNGs) (Bregnocchi et al., 2016). The antimicrobial activity observed in the composites with GNPs and ZNGs nanofillers was much better than in composite with ZnO-NRs. The nanostructure formed by GNPs and ZNGs provided a greater interacting surface with bacteria resulting in good antibacterial activity. PVDF/HAP film was prepared using the solvent casting method to investigate its cytotoxicity for potential application in repairing bone defects (Braga et al., 2007). The cell viability revealed that the composites are noncytotoxic. The mechanical stability of composites was observed to be reduced by the use of hydroxyapatite (HAP) in the PVDF matrix. These studies established the biocompatibility of the samples, and thus they can have applications in bone and dental restoration. Fluorinated membranes have hydrophobic nature, which impedes the attachment of cells and consequently cell proliferation. A composite of PVDF was fabricated with reduced graphene oxide (RGO) in an attempt to enhance its cytocompatibility (Pei et al., 2015). Human umbilical vein endothelial cells were cultured on membranes of RGO/PVDF composite in order to study cellular proliferation response and adhesion. The composites were observed to be better than the pure PVDF. Furthermore, the incorporation of RGO assisted the conversion of alpha-phase PVDF to beta-phase PVDF, which assisted endothelial cells to secret prostacyclin, having antithrombotic functions. Poly(ethylene glycol) PEG is a polyester that can exist in various molecular weights. It is soluble in several organic solvents and water. PEG has a unique capability to maintain solubility and chemical reactivity after surface functionalization and chemical modifications, demonstrating variable biomedical uses. Also, PEG has the capability to not affect the activity of active proteins of cells while interacting with cell membranes (Harris, 1992). A nanosystem was fabricated with PEG supported by carbon nanotubes in the form of nano-cocoons. To study its efficacy as a drug delivery carrier, it was loaded with curcumin. These nanosystems show no toxicity to blood and also promote the growth of L929 fibroblast cell lines. In a saline medium, curcuminloaded nanosystems dispersed effectively and interacted with C6 glioma brain cancer cells. On the other hand, curcumin alone was not able to penetrate brain cancer cells (Simon, Flahaut, & Golzio, 2019). For application as a wound dressing material, polyethylene oxide and poly(ethylene glycol) dimethacrylate was



CHAPTER 1 Introduction to biomedical polymer and composites

used to fabricate hydrogel film (Haryanto & Mahardian, 2018). When poly(ethylene glycol) dimethacrylate was added to hydrogel, it increased the vapor transmission, mechanical strength, and percentage elongation. The tensile strength improved from 5% to 20%. The water vapor transmission rate was observed to be close to the ideal value for favorable conditions for the healing of wounds. PEG/cellulose scaffolds that were environment friendly, nontoxic, and biocompatible were manufactured. A network of close-grained sheets was noticed when PEG was added to regenerated cellulose. This alteration in scaffolds increased compressive strength by 33 times that of regenerated cellulose (Teng et al., 2018).

1.4.3 Gas-permeable polymeric membranes Polymeric membranes have extensive application as oxygenators or for hemodialysis that are used in cardiac surgery in infants, treating underdeveloped lungs and chronic problems. Reduced gas efficiency is found for gas-permeable membranes because blood components (proteins, platelets) have a tendency to get deposited over their surface (Kolobow, Borelli, & Spatola, 1986). 2-Methacryloyloxyethyl phosphorylcholine (MPC) copolymer was prepared to resolve the aforementioned issue by surface modification of conventional polymers. Substrate polymer was coated with MPC copolymers and alkyl methacrylate, which resulted in inhibition of cell adhesion. Also, when blood lacking anticoagulants was put in direct contact with the polymer, the protein adsorption decreased (Ishihara et al., 1992). An oxygenator membrane made up of poly(MPC-co-dodecyl methacrylate) (PMD) skin film adhered to polyethylene (PE) was fabricated (Iwasaki, Uchiyama, Kurita, Morimoto, & Nakabayashi, 2002). This showed that the gas permeability improved with the presence of MPC in the polymer film. PMD/PE membrane under oxygen gas permeation analysis showed similar results in comparison to PE membranes besides the fact that MPC in PMD was more than 0.2 unit mole fraction. It was also observed that there was a significant decrease in protein adsorption on the surface of PMD in comparison to the PE surface. The membrane surface decreases the protein adsorption as the surface restricts the hydrogen bonding with water (Lu, Lee, & Park, 1992). With the help of plasma-induced surface modification, polysulfone (PSF) membranes were fabricated to investigate its application as an artificial lung (Wang et al., 2016). The steric hindrance and surface hydrophilicity of the PSFPEG-Heparin (Hep) membrane was improved, which led to the decrease of adsorption rate of fibrinogen and bovine serum albumin in contrast to pure PSF. However, pure PSF showed better adhesion of platelets. On the other hand, the PSF-PEG-Hep membrane exhibited a steep decline in platelet adhesion with the increase of the molecular weight of PEG. Therefore good platelet adhesion resistance was observed in PSF-PEG10,000-Hep and PSF-PEG6000-Hep. Furthermore, exceptional gas exchange performance was shown by the PSF-PEGHep membrane in the presence of porcine blood. To examine its use as membrane

1.4 Polymers and their composites for biomedical applications

oxygenators, the PSF-PEG-Hep membrane was synthesized utilizing PSF chloromethylation, PEGylation, and heparin immobilization process (Zheng et al., 2016). The blood oxygenation results for the same were at par with the commercially available membrane oxygenators indicating its potential application as an oxygenator in treating various lung diseases. For use as an extracorporeal membrane oxygenator, PSF membranes were fabricated through surface modification with low-temperature plasma treatment. Surface modification of PSF was performed with three additives, Acrylic acid (AA) with heparin (Hep), MPC, and Col. (Zheng, Wang, Huang, Fan, & Li, 2017). PSF-AA-Hep showed the least protein adsorption with an increasing trend, followed by PSF-MPC, PSF-Col, and pure PSF. The same trend was followed for platelet adhesion. It is expected that charged groups from heparin exhibited steric hindrance, which restricted the protein adsorption. Similarly, biomimetic structures from Col or MPC, and hydrophilic groups from AA were the reason behind the trend followed by these membranes. Gas permeation activity of surfacemodified PSF membrane was low as compared to pure PSF, as its surface was covered with grafted molecules. Thus the study showed that a modified PSF membrane could be potentially applied as a membrane oxygenator or in artificial respiratory devices (Zheng et al., 2017).

1.4.4 Other polymeric composites Polymers and ceramics are the most widely used materials in biomedical applications (Holzapfel et al., 2013). But they individually do not meet all the basic requirements like biocompatibility, mechanical load bearing capability, corrosion, and wear resistance. So researchers are developing composites with polymer matrix reinforced with ceramic to introduce bioactive characteristics of the polymer coupled with mechanical strength and corrosion resistance of ceramics (Dziadek, Stodolak-Zych, & Cholewa-Kowalska, 2017). A study was conducted by Gil-Albarova et al. (2012) to evaluate the in vivo performance of cross-linked glutaraldehyde, gelatin-coated nanocrystalline hydroxyapatite binding peptide (HABP) scaffolds. HABP foam cylinders were implanted at the site of artificially created bone defects in the femurs of New Zealand rabbits. The foam was found distended after being filled with bodily fluids, thereby ensuring proper fixation without cementation. In the histological and radiological studies conducted after 4 months, it was found that the implantation treated the critical-sized bone defect with osseointegration and osteoconduction at the surface of the foam, illustrated in Fig. 1.10. The results indicate that a gelatin-coated hydroxyapatite scaffold has the potential to be used for clinical conditions in orthopedics and dentistry (GilAlbarova et al., 2012). 3-D printed biomimetic Col/hydroxyapatite (CHA) composite material scaffolds were prepared using a low-temperature additive manufacturing technique (Lin et al., 2016). The CHA scaffolds facilitated the proliferation of bone marrow stromal cells and enhanced the osteogenic response in vitro. The scaffolds were



CHAPTER 1 Introduction to biomedical polymer and composites

FIGURE 1.10 (A) Macroscopic image of the femoral bone specimen of the rabbit after necropsy, (B) histological staining image illustrating the integration of bone tissue (indicated with black arrows) in the HABP foam cylinders; Masson’s trichrome staining images illustrating: (C) formation of new bone tissue in the form of trabeculae (indicated with a white star) around the foreign material (shown with black arrow) and bone marrow (shown with black asterisk), and (D) membranous ossification process (indicated with black wavy arrow) with newly formed bone tissue (shown with white star) around the foreign material (black arrow). Adopted from Gil-Albarova, J., Vila, M., Badiola-Vargas, J., Sa´nchez-Salcedo, S., Herrera, A., Vallet-Regi, M. (2012). In vivo osteointegration of three-dimensional crosslinked gelatin-coated hydroxyapatite foams. Acta Biomaterialia, 8, 3777 3783, with permission from Elsevier.

implanted in defective femoral condyles of rabbits to investigate the in vivo performance of the scaffolds. The scaffold constructed with 600 μm diameter rods was found to have optimal mechanical stability and, prior to degradation, promoted cell penetration and mineralization along with improved healing of the bone defects. The results indicate that 3-D printed CHA scaffolds are suitable for applications in tissue engineering and regenerative medicine. In a recent study conducted by Alonso-Sierra et al. (2017), biomimetic inorganic/organic composites were fabricated, and the porosity analysis of the same was carried out using X-ray microtomography. Hydroxyapatite was prepared by gel casting method and was molded into the 3-D hierarchical interconnected porous structures by adding PMMA microspheres. The organic matrix was burned off during sintering to achieve a controllable porosity of the hydroxyapatite

1.4 Polymers and their composites for biomedical applications

scaffolds. Finally, two organic phases, gelatin, and Col were used to generate an organic inorganic composite material, after extraction from the bovine tail. The compressive strengths of the gelatin and Col-based composites were found to be roughly three times that of the natural bone tissue. The achieved pore size distribution will facilitate cell proliferation, ingrowth of intermediate tissue, ensure vascular incursion, and nutrient supply. The study showed that this composite is a promising material to substitute bone tissue and can be used in the manufacturing of prostheses. Recently, a study evaluated the cytotoxicity in vivo and antibacterial behavior of a PCL/hydroxyapatite/gelatin scaffold loaded with doxycycline. The antibacterial study showed that the codelivery of hydroxyapatite nanoparticles along with doxycycline inhibited the bacterial growth of Gram-positive S. aureus and Gramnegative Porphyromonas gingivalis bacteria. In vitro doxycycline release profile in the phosphate buffer medium was characterized by two steps. Initial burst release of the drug from the scaffold was found to be 60% within an hour, followed by a continual release of the remaining drug for 55 h. The anticancer activity of the scaffold was studied by testing the sensibilities of three cancer cells (A-431, 4T1, and CACO-2) to the codelivery system. It was found that the A-431 exhibited the most synergistic effect compared to 4T1 and CACO-2 cells. Based on the anticancer and antibacterial results obtained in this study, the developed doxycycline-loaded PCL/hydroxyapatite/gelatin composites were found suitable as a drug delivery system (Ramı´rez-Agudelo et al., 2018). A three-component poly(lactic acid) (PLA)/PCL/wollastonite material system was fabricated to evaluate their suitability as tissue engineering scaffolds (Goswami, Bhatnagar, Mohanty, & Ghosh, 2013). The porous foams prepared from the composites were tested in vitro for biocompatibility by seeding in osteoblast cells. The prepared foams exhibited enhanced proliferation and fashion of osteoblast cells over the 7-day incubation period. The materials were also tested under compressive loads in dry and wet conditions for simulating their performance in physiological conditions. Increasing the wollastonite content led to an improvement in hydrophilicity of the polymers, and that facilitated better implant tissue adhesion. It was found that composite (PLCLW8) containing the highest amount of CaSiO3, facilitated cell adhesion and proliferation at a faster rate compared to the pure polymer (PLCL15). In a study conducted by Bheemaneni, Saravana, and Kandaswamy (2018), cytotoxicity and mechanical properties of poly(butylene adipate-co-terephthalate)/ wollastonite biocomposites were evaluated. The composite exhibited good formation of hydroxyapatite layer after being soaked in simulated body fluid within 5 days. Increasing the wollastonite content led to enhancement of the tensile strength of the composite. The composite facilitated enhanced MG63 cell proliferation within a short incubation period. It was found that adding the bioactive silicate filler (wollastonite) helped in biomineralization as well as cell proliferation compared to the pure polymer [poly(butylene adipate-co-terephthalate)] (Bheemaneni et al., 2018).



CHAPTER 1 Introduction to biomedical polymer and composites

Recently, hydroxyapatite/PLLA electrospun membranes were fabricated to study their potential applicability as bone substitutes (Santos et al., 2017). The biocompatibility of the hybrid composite membranes was studied. Results of in vitro MG63 osteoblast cell culture suggest that the membranes promote better cell proliferation compared to PLLA membranes, and processing methods do not induce any cytotoxic effect in the cells. The results of the metabolic activity indicate a faster rate of cell growth on the surface of the hydroxyapatite/PLLA when compared to PLLA and the control membranes. The simple and scalable processing method used makes the fabricated polymer microparticles fiber membranes a potential bone substitute. In a recent study, thin PLA composite films reinforced with coralline hydroxyapatite were fabricated. The coralline hydroxyapatite content of the composite promoted human adipose-derived stem cell attachment and proliferation, contrary to no cellular activity seen on the surface of pure PLA. The bioactivity and osteoconductivity of the ceramic, in addition to the flexibility and biodegradability of the polymer matrix, make this composite a potential biomaterial for designing scaffolds (Macha et al., 2017). Hydroxyapatite/ultra-high molecular weight PE composites with different ratios of hydroxyapatite were prepared for evaluating the role of the ceramic filler in biocompatibility (Mirsalehi, Sattari, Khavandi, Mirdamadi, & Naimi-Jamal, 2015). All the samples facilitated enhanced adhesion and proliferation of the MG63 cells compared to the control material. The results obtained indicate that the composite with higher hydroxyapatite content exhibited better bioactivity due to enhanced differentiation and proliferation of osteoblast cells. It was concluded that composites having polymer matrix reinforced with bioactive ceramic fillers have potential applicability as nontoxic biomaterials that can initiate bone ingrowth on their surface.

1.5 Challenges and future trends The use of polymers and polymeric composites for fabricating new biomaterials and improving the existing biomaterials is gaining a lot of traction in the biomedical industry. In a nutshell, this can be attributed to the broad spectrum of mechanical and biological properties exhibited by the polymer and polymer composite biomaterials. The composite biomaterials can be tailored for designing implants with an overall structure and interface properties that can elicit favorable responses from the surrounding tissues. Despite this, the present areas of application of these biomaterials in medicine are significantly lower than what was extrapolated a few years back. In many cases, although the biomaterials showed a lot of potential in the research and development stage, they were not included in the production and subsequent commercial distribution of medical devices in any capacity (Salernitano & Migliaresi, 2003). The major critical issues hindering the use of these materials are summarized here.

1.6 Conclusion

The experimental and clinical data supporting the long-term applications of composite materials are remarkably less compared to the same for homogeneous materials. One positive aspect of composites is the controllability of material properties by varying the constituents and the type and orientation of the reinforcement phase. This makes composite suitable for designing implants with optimized adaptability to the mechanical properties of the host tissue (Chung, Im, Kim, Park, & Jung, 2020). However, the additional design parameters make the fabrication process more complicated. An ideal biomedical scaffold should possess excellent biocompatibility, must be fully resorbable, have mechanical stability, can be easily molded into the required shape tailored to the implantation site, and have an internal geometry that facilitates tissue ingrowth and ensure proper vascularization (Palmieri, Sciandra, Bozzi, De Spirito, & Papi, 2020). The lack of satisfactory standardized tests for evaluating the biocompatibility of composites makes it difficult to comment on the suitability of a composite biomaterial scaffold in a particular clinical setting. Fatigue testing of a material is important for determining its use in an implant as in vivo loads are predominantly repetitive or cyclic in nature. There is no adequate standardized testing available for assessing the performance of composite materials under fatigue loads. Moreover, compared to the monolithic materials, determining the fatigue behavior of composite is much more complicated. Overcoming the challenges mentioned here is integral before polymer composites can be extensively used in the biomedical industry. However, the future prospects for the widespread commercial use of composite biomaterials in medicine are very promising. 3-D printing and advanced manufacturing techniques have to facilitate the fabrication of scaffolds with the desired porous network without the use of any cutting tools, molds, or dies. Hence, this can be used to construct patient-specific scaffolds that are compatible with the mechanical properties of the neighboring tissue at the implantation site (Nakayama, Shayan, & Huang, 2019). Composite materials might also find applications in other clinical areas where materials with new properties are needed for developing more efficient alternative devices and surgical techniques.

1.6 Conclusion In this chapter, a detailed review of the recent developments in fabrication techniques and biomedical applications of polymers and polymeric composites has been reported. From the recent trends, it can be observed that the composite approach for developing new biomedical scaffolds and modifying existing materials is becoming more and more important in the field of biomaterials. This can be attributed to the tailorable manufacturing of composite materials and properties comparable to the host tissue. The successful development of novel polymers and polymeric composites targeting particular medical applications depends upon the



CHAPTER 1 Introduction to biomedical polymer and composites

firm grasp of the fabrication technique, material properties of the component materials, and a thorough understanding of biology and medical science. Based on such a foundation, new and innovative polymer and polymeric composites can be tailored and fabricated for a variety of applications in biomedical engineering. However, for the application of composite materials in a clinical setting, medical professionals need to be convinced about their long-term durability and reliability. Compared to monolithic materials, the experimental and clinical data supporting polymer and polymeric composite biomaterials are relatively small. Thus further research into polymers and polymeric composites can elucidate their long-term durability under physiological conditions. The successful development and applications of polymer and polymeric composites demand harmonious cooperation between the healthcare and engineering industries.

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CHAPTER 1 Introduction to biomedical polymer and composites

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CHAPTER 1 Introduction to biomedical polymer and composites

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Foundation of composites


Umesh Kumar Dwivedi and Neelam Kumari Department of Physics, Amity School of Applied Sciences, Amity University, Jaipur, Rajasthan, India

2.1 Introduction For several years composites have been used to solve technical problems. Polymer-based composites were introduced in the 1960s and more industries began to take notice. Since then, composites have become a standard engineering material. The increase use is due to the product performance and their light weight components (Pal, Jit, Tyagi, & Sidhu, 2011). Composites are composed of two or more distinct materials called constituent materials. These constituents are not chemically bonded together and have different properties. It is the combination of different constituents that result in superior physical properties. The constituent phase that is continuous throughout the composite is termed the matrix. The particles/fibers which are dispersed in the matrix making it stronger are called the reinforcements. Processing of composites is affected by both the reinforcements and the matrix. The main function of the matrix is to carry load, maintain the reinforcement fibers in proper orientation, and bind the reinforcements together. It distributes the load throughout the reinforcements through interface and protects them from environmental damage. The reinforcing phase provides the toughness and strength. The matrix material may be metallic, polymeric, or ceramic, and reinforcement may be fiber or a particulate. Particulates in a composite should be evenly distributed. The reinforced particulate composites are less expensive, less rigid, and much weaker as comparable to fiber-reinforced composites. The particulate composites have disadvantages such as brittleness and processing difficulties when the particulate/matrix volume percentage is more than 30% 40%. Fibrous composites are formed by stacking single sheets of continuous fibers into laminate to obtain the desired rigidity and strength and can include 60% 70% of fibers. Due to small diameter of fibers, composites are very strong. Examples of fibers include carbon, glass, and Kevlar. Final properties of composites depend on the type of matrix and reinforcement. Continuous and discontinuous fibers have different properties. For instance, continuous fibers have greater strength and rigidity, whereas discontinuous fibers are random in orientation. This random orientation tends to decreases the strength and rigidity of the final Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 2 Foundation of composites

product. Continuous fibrous composites are used where high strength and rigidity are required. Discontinuous fibrous composites are used in low strength and rigidity applications.

2.2 Classification of composites On the basis of the matrix phase, composites are divided into polymer, ceramic, and metal (Malhotra, Goda, & Sreekala, 2012). Three main types of polymer used as matrices are thermoset, thermoplastic, and elastomers as shown in Fig. 2.1. Once cured, thermosets cannot be melted. Thermosets have good affinity to heterogeneous materials, for example, epoxy, polyester, etc. The most common resins used in thermoset composites are epoxy, phenolic, and polyester. Thermosets have the advantage over thermoplastic at high temperature and creep resistance. On the other hand, thermoplastic materials are ductile and tougher than thermoset materials. Thermoplastic are reversible in nature, that is, they can be reshaped by the application of pressure and heat. Thermoplastic molecules are flexible and reformable into previous shape due to the cross-link behavior. Some thermoplastic resins are polypropylene, polythene, etc. The term “elastomer” stands for elastic polymer and is frequently interchangeable with the term “rubber.” Elastomers have both viscous and elastic properties, generally having low longitudinal stress-to-strain ratio and high yield point as compared to other materials. Polymers are made up of monomers formed mostly from the elements of carbon, hydrogen, silicon, and oxygen. Examples of polymers are polyvinyl chloride, polyethylene, etc. Elastomers exhibit no definite form above 370 C. Natural rubber, polybutadiene, butyl rubber, etc. are some examples of elastomers. Two types of matrices are mainly used for fabrication process, that is, thermoset and thermoplastics. An inflexible solid will be formed by processing above melting temperature and curing the thermoset resin. Since the resin is intractable, it cannot be melted and remolded into a different shape, whereas thermoplastic can be remolded to new shape by heating the previously cured shape. These polymer composite matrices are favored over other materials due to their undemanding fabrication, greater strength-to-density ratio, and corrosion

FIGURE 2.1 Types of polymer.

2.3 History of composites

FIGURE 2.2 Classification of composites.

resistance. They can be classified on the basis of different reinforcements as shown in Fig. 2.2. Composites with a polymer matrix can be used where the addition coatings such as oil and grease cannot be tolerated. Different polymer possesses different properties such as biocompatibility, biodegradibilty, and high mechnical strength which define their uses in appropriate applications. These matrices are easily processed, are low in cost, and have low relative density. On the other hand, low rigidity, low strength, and low transition temperature limits their use. These requirements can be attained by combining nonmetal reinforcements in the polymer matrix. Using metal reinforcements, natural or synthetic fibers, and ceramic particles are often employed in a polymer matrix. Metallic fibers have limited use due to their high density and reaction affinity with the matrix. Due to excellent properties of polymer composites, these are extensively useful in medical fields such as tissue engineering, dentistry, implantation, prosthesis, etc. The biomedical materials are basically a combination of natural fibers and polymer matrix. These composites have excellent biocompatibility and match the morphology of a living being. The classification of biocomposites, fabrication techniques, and its biomedical applications are discussed later in this chapter.

2.3 History of composites In ancient times builders and artisans reinforced mud with straw making bricks. Around 1500 BCE strong and sturdy buildings made up of these bricks were used



CHAPTER 2 Foundation of composites

by Egyptians and Mesopotamian settlers (Scala, 1996). Pottery and boats were reinforced in this manner, as well. The Mesopotamians were first credited for use of composites in the form of plywood, created from glued woods. Later, composite bows were invented by the Mongols in 1200 CE. These bows were made of the combination of wood, bone, bamboo, and horns bonded with animal glue. These bows were extremely deadly weapons on the Earth during that time. Mongolian bows provided the emperor with military dominance until the invention of gunpowder in the 14th century. In Indian history, women contributed to the field of composite by reinforcing cow dung with hay to make cow dung cakes. These cow dung cakes are still being used today to cook food in villages. The fashionable era of composites did not fully begin until scientists developed plastics. In 1869 a chemical revolution changed the composite revolution. People started to experiment with resin. These resins completely transformed from liquid to solid state. Glue and binders were some natural resins obtained from plants and animals. Within the early 1900s plastics such as vinyl, polystyrene, phenolic, and polyester were created. These plastic materials were far better than natural resin. However for better strength and rigidity, reinforcements were required. Fiberglass was introduced by Owens Corning in 1935. An incredibly strong, light weight structure was created by combining fiberglass with plastic polymer. This is the start of the fiber-reinforced polymer (FRP) industry as we all know it today. World War II brought the need for light weight, strong, and weather-resistant composite materials and the production of FRP began. Before WWII, aircraft wings consisted of wood and plastic resin. Aircrafts used during WWII were light weight and corrosion resistance as they consisted of fiberglass. By 1947 an automobile of fully composite body was tested, which led to the manufacture of 1953 Corvette. Fiberglass was employed in pipes on a commercial scale and later adopted by the refining industries. In addition to light weight and strength of fiberglass, researchers learned that fiberglass was transparent to radio frequency (RF) bands. Fiberglass was quickly adaptable to electronic radar equipment during the war. By WWII, military wanted fiberglass instead of FRP composites.

2.3.1 Fiberglass in 20th century In the 20th century the world of automobiles and boats fully depended on fiberglass composites. After 1950s the outer body of boats were reinforced with fiberglass materials. After this, fiberglass wakeboards were introduced (Sharma, Bhanot, Singh, Undal, & Sharma, 2013). During this period, classic cars were manufactured by the automobile industry using fiberglass impregnated with resin and molded with fine-shaped dies. In 1953 a new composite Chevrolet Corvette was manufactured. These composites are carbon fiber-derived composites and proved to be slug- and knife-resistant, upgrading safety for the military and police forces.

2.5 Advantages of composites

2.3.2 Composite material in our daily life In the present, composites are employed in the technology of our daily life, for medical supplies, and to the department of defense. Nowadays automotive and aerospace fields are fully dependent on composite technology. Military and police forces utilize these new composites in gunpowder, firearms, automobiles, and aircrafts. Some composites are very light weight and this opens diverse applications in the medical field and equipment technology, etc. National parks are using composites to rebuild foot and vehicle paths and bridges without the need to demolition natural surroundings or use heavy machinery. As technology in composite manufacturing grows, this draws our attention to sustainability and the composites effect on the environment.

2.4 Why composites? The most essential reason to use composite materials is the adaptability of their properties. Composites are chosen due to their improved rigidity and strength while being light weight and to increase acceleration or range in transportation. Composites can be used as fire and blast protection as they are thermal insulators and are also used in parts of machinery. Other reasons to use composites in various applications are durability, strength, rigidity, stiffness, and corrosion-resistant properties. Nowadays, composite materials are used in large number of engineering fields such as aviation, automobile, and robotics. Composite materials have certain strengths and weaknesses which should be taken in consideration. However, the major driving force that is responsible for development of new composite materials is the many combinations of reinforcements and matrices possible to meet the requirements of applications. Composites can be engineered and produced to be sturdy and hardy in a particular alignment, whereas metals are of same strength in all directions. The sheer repertoire of the composites is mind boggling.

2.5 Advantages of composites 2.5.1 Design flexibility Designers can experience unlimited flexibility while designing different shapes from thermoset polymer-based composites. They can be formed into the most intricate shaped components and be made in a large range of densities and chemical formulations to precise performance specifications. Molding of composite in any shape is easier than other materials. Fiberglass composites are materials which can easily form any complex shape. Most recreational boats are built from fiberglass composite material, lowering the cost of boats and improving the boat



CHAPTER 2 Foundation of composites

design. The surface of the piece can be finished with a varity of textures, smooth to pebbly.

2.5.2 Light weight Composites are light weight, collated to various metals and woods. The light weight of composites are vital in automobile and aircraft industries. For example, light weight materials result in better fuel efficiency. While designing airplanes, the weight of the craft is always considered by the engineers; reducing the aircraft’s weight reduces the amount of fuel it needs as it acquires more speed. More composites are employed to build modern aircrafts than metal. The new Boeing 787 Dreamliner is made up of composites (Giurgiutiu, 2015).

2.5.3 High strength Aluminum and steel are weaker in strength in comparison to composites. Metals have equal stiffness and strength in all directions, whereas composites have strength in a specific direction in which it required.

2.5.4 Strength related to weight The strength-to-weight ratio can be used when deciding which composite to use for a specific application. Steel itself is a very rigid, heavy material. Other material may be strong and light weight, like bamboo poles. Composites are often styled to be both firm and light weight. Because of these features, composites are used to manufacture airplanes which need a highly compact material on the underneath side. Composite materials exhibit excellent properties in one direction due to unidirectional alignment of fibers in matrix. For example, composites made with metal fibers that must be in an alignment, require more precise processing. But the metal used is often heavy, which adds weight to the finished composite. Composites have strength without being substantially heavy. Composites that have higher strength-to-weight ratios are in structures today.

2.5.5 Corrosion resistance The matrix in composites protect the reinforcement materials from seasonal damage and harmful chemicals that can damage other materials. Composites are better choice for protecting chemicals in stores and outdoors. Composites can stand up in bad weather conditions and during temperature changes.

2.5.6 High-impact strength Many composites can absorb impacts, blast from an explosion or sudden force of bullet. Because of this unique property, bullet-proof vests and panels are made by

2.5 Advantages of composites

using composites. Airplane, buildings, and military vehicles can be shielded from explosions by composites.

2.5.7 Consolidation of many parts Whole assembly of metal parts can be replaced by a single composite materials. Time can be saved by using less number of parts or components in machinery. Less number of parts used means less maintenance needed for a machine.

2.5.8 Dimensional stability Composites are free from temperature effect. Size and shape of composites can remain the same in any weather condition. As the weather changes to hot or cool, wet or dry, swelling and shrinking of wood takes place. For example doors of houses that are made of wood, swell and shrink during weather changes. In rainy seasons wood doors swell and more force is required to close the doors (Selzer & Friedrich, 1997). A situation where tight fit is required, composites can be a better choice. Composites are used in the wings of aircrafts, as the plane gains altitude, size and shape of wings do not change.

2.5.9 Nonconductive Many composites are nonconductive, they do not conduct electricity and are insulators. Electrical utility poles and the electric circuit boards in electronics are made with composites. However, conposites can be made conductive by using conductive filler reinforcement in matrix.

2.5.10 Nonmagnetic Composites are nonmagnetic as they contain no metals in matrix. Any metal reinforcements are shielded by the matrix. They can be used around delicate equipment. The lack of magnetic intervention from the composites used in the housing allows bulky magnets utilized in magnetic resonance imaging machine to perform better. Furniture is also made up to composite material. Floors and concrete walls of hospitals are made from reinforced concrete. In a whole, nonmagnetic nature of composites helps in development of infrastructure of houses and buildings.

2.5.11 Radar transparent Composites are radar transparent materials, which means that radar signals to pass easily through the material. This property of composites makes it a suitable material to use anywhere, whether the radar is on ground or in the atmosphere. The US Air Force’s B-2 spy plane is made up of composite, which is nearly invisible to radar. Radar signals cannot capture the stealth aircraft.



CHAPTER 2 Foundation of composites

2.5.12 Low thermal conductivity Composites do not conduct heat or cold easily as they are good insulators. Door, panels, and windows of buildings that are made from composites work as extra protection in severe weather.

2.5.13 Durable Structures made with composites are long-lasting, very durable, and need little maintenance. Many composites have been in use for 50 years.

2.6 Applications of composites Today, thermosetting composites are employed in a vast range of industrial applications that have been described in the following sections:

2.6.1 Aerospace/aircrafts Wings, fuselages, and bulkheads are some applications in aerospace which are made of thermoset composites for commercial, military, and civilian use.

2.6.2 Appliances Thermoset composites have wide range of application in the appliance industries, such as washing machine, hair dryers, refrigerators, microwave ovens, freezer panels, electric controls panels, handles of cookware, power tools, side trims, and vent trims.

2.6.3 Automobile and transportation Composites are widely employed in automobile industry and in transportation. Composite materials are light weight and strong. Due to these properties, they are used for the internal components of cars, buses, and trains and also the outer body parts.

2.6.4 Infrastructure Composites are being used in infrastructure applications such as in buildings, bridges, and roads. Strength and durability of composite material are the major properties designers want in the material that we use in our daily life. Thermoset composites are used in construction, replacing many traditional materials. In homes, doors, wall panels, fixtures, roofing, sinks, shower stalls, etc. are architectural components that can be replaced by thermoset polymer composite with

2.7 Limitation of composites

some additional reinforcement. These composites are having far better result in comparison to traditional materials—aluminium and steel.

2.6.5 Environmental Composites are often seen in applications in corrosive environments. These materials are said to be ideal for many types of application, such as natural resource refineries, chemical processing plants, water purification and treatment facilities, etc. Some of the ordinary things that are made of thermoset materials are tanks, hoods, pumps, pipes, fans, and cabinets.

2.6.6 Applications of electricity Composite materials have many applications in electrical industries due to their high dielectric permittivity, good arc resistance, and scratch resistance etc. Examples of components made of these composites are microwave antennas, wiring boards, substation equipment, motor controller, standoff insulators, control system components, circuit breakers, arc chutes, terminal boards, metering devices, gear switch, bus supports, and lighting components.

2.7 Limitation of composites Some of resins used in composite materials can withstand a temperature of 150 C or less, which means the resin will weaken at 140 C 150 C. This may increase the risk of fires in aircrafts or vehicles. Fires within composite materials can spread toxic gases and microparticles in atmosphere, causing health hazards. Temperatures above 300 C can result in structural breakage. Some composites are very brittle in nature and due to this, these materials can be easily broken (Lau, Ho, Au-Yeung, & Cheung, 2010). Composites involve such assembly where out-of-plane direction of reinforcement leads to poor strength of composites. The primary load is carried out by matrix which impacts mechanical susceptibility and requires more careful repairing as compared to structures made of metals. Due to brittle characteristic of some composites, they easily get damaged as compared to wrought metals. Cast metals also have some brittle character. Metal reinforced composites have low shelf lives because of oxidation of metal with time which introduces corrosion, abrasion, etc. and require refrigerated transport and storage. Some special equipment is required to hot curing. Curing process takes time for heating and cold. Pressure and tooling is required to repair at original cure temperature. Cleaning of composite should be done before repairing to make them free from all contaminates present. Some fibers and matrices absorb moisture that leads to the need of the composite to be dried at a particular temperature before repair.



CHAPTER 2 Foundation of composites

2.8 Biocomposites and classification 2.8.1 Biomedical composites It is worth addressing that the most noteworthy advancements in the field of composites are biomedical ones. Biomedical composites are used to supplement or replace tissue in the human body. Several uses of biomedical materials were noted from primeval civilization. Artificial nose and eyes were found on Egyptian mummies. For the repairing and reconstructing the defective parts of body, People of India generally use biomaterials such as wax, glue, etc. Some of these biomaterials are useful today for the purpose of cleaning and shinning household furniture. Over the centuries, advancement in many composite materials has promoted bioresearch in many ways such as medical practices these days. Medical practice these days utilizes biomedical composites in several applications, such as limb replacements, joint replacements, and dental implants (Pye, Lockhart, Dawson, Murray, & Smith, 2009). Medical devices, such as biosensor pacemaker, artificial hearts, blood vessels, or organs, are widely used to supplement and bring back the function of traumatized and degraded tissues to help in healing and improving function, thus increasing the life span of patients. Biomedical materials and biocomposites are fabricated to operate with the body.

2.8.2 Basic requirements and parameters for biomedical applications Biocompatibility Biocompatibility is the ability of graft material to function without damaging the systematic response of the body. Prior to use, the biomaterials first undergo toxicity and compatibility testing to determine the safety. Some materials such as nickel and chromium can cause allergic reactions to skin or tissue. Corrosion Corrosion is oxidation deterioration of metals. When the metal corrodes that means it will weaken the mechanical property of metals and this will weaken the chance for complete bone healing. Several types of corrosion could take place during orthopedic surgery to repair broken bones. Most common corrosion is galvanic corrosion. In galvanic corrosion metals which are in electrical contact with one another undergo corrosion. The metals are immersed into an electrically conductive medium, that is, in human body. The corrosion not only harms the surface of the orthopedic grafts but also acts as stresser which damages other soft tissue attached to it. Mechanical properties Mechanical properties must be kept in mind while designing a bone fixation device or an implant. Design of implanting device required attention to size,

2.8 Biocomposites and classification

structure, long time stability, and mechanical properties. Mechanical properties depend on a number of factors, such as the wear resistance properties under stress and strain from use, how it shows fatigue, it’s viscoelasticity and isotropic nature. For example, in dental prostheses, silicon carbide/carbon fibers composite have low elastic modulus with appropriate strength with that of natural teeth which reduces the chance for stress, giving replaced teeth a better lifetime (Ramakrishna, Mayer, Wintermantel, & Leong, 2001). Pores Pores are defined by several measurments, such as degree, size shape, and interconnectivity, and are a essential part of the biomedical application. Low-cost titanium-based composites are porous in nature to provide interconnectivity with the cells it is placed with. Titanium rods do not harm the soft tissues of body. Eye glasses Composites have replaced the glass used in spectacles, as well as being used for the frames. The plastic glasses are stronger and lightweight compared to normal glass. The raw material used for plastic glasses is cellulose acetate which is strong and flexible. The embedment of certain particulate such as nickel and titanium makes the glass mechanically more stronger. These glasses can be coated with a scratch resistant film. Biodegradability and bioabsorbable polymer Biodegradable polymer are used for various applications in medical fields such as surgical sutures, drug delivery system, bone fixation, aid in cell adhesion, drug carrier, vascular grafts, artificial skin, etc. These polymers are biologically decomposable and do not harm the cells in body. High cell adhesion and less inflammation In many biological applications controlling surface to cell adhesion is very important parameter. Cell to surface adhesion is a crucial factor studied in tissue growth research which indicates whether cell culture could develop into functional tissue. Biomedical composites must have high cell adhesion properties. In medical implantation, adhesion between implanting device and peripheral tissues is of enormous value to make implants successful. It must be kept in mind that implanting a medical device should create less inflammation to the surrounding tissues. Wear resistance In biomedical composite applications such as in dentistry, bone graft, and bone cement, the wear resistance behavior of composites is very important. Wear causes less stability and lifetime of joint or dental implant. High wear resistance properties of some polymers such as polyurethane, calcium phosphate, polyethylene terephthalate, etc. enhances the long-term stability of planted device. On the



CHAPTER 2 Foundation of composites

other hand, proper alignment of reinforcement fillers in the polymer matrix improves the mechanical properties and wear resistance of the replaced part. Wear debris is minimized by incorporating fillers in composites.

2.8.3 Biomedical polymer composites Biomedical polymers are macromolecular compounds obtained from natural origin. Biomedical composites are flexible and are resistant to chemical attack. These composites have good compatibility with body. There are a wide variety of biomedical composites available with different physical and chemical properties. These materials are easily moldable in any shape. Biomedical composites are classified into two types, depending on their origins: • •

Natural biomedical polymer Synthetic biomedical polymer Natural biomedical composites Natural biopolymers are derived from living organisms and easily replace injured tissue. Biocomposites have stimulated, or prompted researchers as it is the best eco-friendly alternative on the market. Fiber reinforced polymer is made of a polymer matrix reinforced with fibers (Bharath & Basavarajappa, 2016). Natural fibers are usually extracted from plants, such as pineapple, hemp, etc. Several varieties of plants are currently in use and more are being researched. Plant fibers have proven their position in the field because their properties are absolutely remarkable compared to other sources of fibers. These fibers have low density, are renewable, biodegradable, highly porous, and available in large quantities. Their mechanical and physical properties make them better than other types of composites available on the market. Some fibers are obtained from living plants, however some fibers can be extracted from the waste of the crop. Fiber properties vary from one plant to plant, but the properties also vary depending on the harvesting methods, crop management, and the treatments the plant undergoes. Biocomposites are eco-friendly, however, many different industries are researching to find out whether they are completely biodegradable. An epoxy matrix, that is not biodegradable, with fibers from the kenaf plant produces a partially biodegradable biocomposite. When designing a biodegradable biocomposite, it is important to select a biodegradable matrix. Polylactic acid (PLA) is a biopolymer and widely available in the market however, it is appropriate and is one option present in the market; however, it is appropriate only for those applications that do not require high mechanical performance at high temperatures or do not need long-term durability. Other options available in the market are polyglycolic acid, poly-b-hydroxyalkanoates, and polycaprolactone. Natural fibers are sensitive to temperature and must be kept at temperatures lower than 200 C at all times. To satisfy this requirement, matrices having low melting point are needed. Due to

2.9 Applications of biocomposites

its lower mechanical strength, these polymers are rarely used. Thus these biocomposites have limited applications. Synthetic biomedical composites Advancements in production techniques and material science work together in the production of synthetic biocomposites that play a crucial part in tissue engineering. Composites can be designed or fabricated by using materials such as man-made resin and inorganic materials. The objective of this amalgamation is to maximize the advantages and minimize the drawbacks. Required characteristics can be attained by adding reinforcing inorganic fiber to methacrylate or urethane dimethacrylate synthetic polymers. Poly(methyl methacrylate) (PMMA) is the most suitable polymer for restoration of dentin and enamel. Biocomposites made up of ceramic and glasses are also used in dental tissue regeneration. The embedded inorganic particles are used to overcome inherent weakness of the polymer. Over the past few decades, scientists have made great advancements toward bone and dental tissue engineering by developing porous resin with desired mechanical properties. Some of the synthetic materials or polymers used as biodegradable polymers are: 1. 2. 3. 4.

polyesters polylactides polypeptides polyglutamic acid polyanhydrides

2.9 Applications of biocomposites Applications of biocomposites are many in the medical and dental fields, such as tissue regeneration, wound healing, and even cancer treatments, (Fig. 2.3) and in various parts of human body (Fig. 2.4). Biomedical composites can be used alone and as a complement to standard materials. Biocomposites can be used for improving health and safety in the production of these polymers as they are lighter in weight and environment friendly. In designing biocomposites and anticipating their performance, many issues must be taken into account related to their biological reaction. As the number of scaffold material increases, therefore the host material can be variable. If the reinforcement is biocompatible, the host material, that is, matrix with reinforcement will itself be biocompatible to each other. The biocomposites can be styled in such a way that there will be no interaction of filler particles with the host tissue, but this is demanding because it means eliminating all spaces at the fiber or particle matrix interface throughout fabrication. Bulk form of composite structures directly affects the host material reaction with body part and may lead to serious infection as compared to tiny/particulate form of composite which will not cause severe effects. For instance, ultrahigh-molecular-weight polyethylene is a highly cross-linked, biocompatible



CHAPTER 2 Foundation of composites

FIGURE 2.3 Commercial applications of biomedical polymer.

FIGURE 2.4 Various applications of biocomposites in human body.

2.9 Applications of biocomposites

polymer that has recently become a prominent material in hip replacement as an acetabular cup, whereas fibrous forms of acetabular cup are generally biocompatible but cause more adverse reactions (Ge, Kang, & Zhao, 2011). While nanofillers, whiskers, microspheres are small in size and cause less irritant to human body, the immune cells could envelop the composite and transport it to other parts of the body. This may result in the release of enzymes that could influence the functioning of composites, such as by changing the speed at which degradation is expected. In addition, in dental and orthopedic moving parts, friction increases. New voids at the interface could occur and could cause abrasions to the host. Exposure of the filler particle to the host is also a concern. Therefore the interlinkage of filler material to the host boundary is important for composite performance. The tissue response may affect the material in different ways, such as puffiness of facial spaces with body liquid and fibrous tissue. It should be noted that thermoset polymers are uncommon in tissure implants in human body because they contain nonreacted monomers and cross-linking agents. The size of glass fibers is another important issue to kept in mind during reinforcing in thermoset and thermoplastic polymer. Some glass pieces may also percolate from the matrix if they are not fully removed during processing, but this is not a concern if the application is on the outer part of body. Following are a few examples to illustrate the use of composites in the medical and dental fields.

2.9.1 Tissue engineering The main purpose of biomedical composites in tissue engineering is to regenerate the damaged tissue. The biocomposites act as a template or scaffold to support the growth of new cells and are cell- and tissue-specific materials that help the tissue integration (Seal, Otero, & Panitch, 2001). Cells attached through biodegradable polymer composites have smaller dimension as compared to actual cells available in body. Repairing the blood vessels and healing the wound at the same time is a major requirement in the medical science. Bone of human body is considered composites of protein having a different morphology such as a variety of shape, structure, and size. These composites consist of hydroxyapatite (HA) nanocrystals that are deposited along the collagen fibers in bone. Collagen fibers have low elastic modulus. These fibers are oriented in the direction of stress. About 70% of dry bone weight can be covered by HA. According to Wolff’s law, bone can remodel and adapt itself according to applied mechanical surroundings. Depending on the location in the body, there are different kinds of fracture. To attain proper fixation of bone, it is required to follow the proper procedure of implantation with control development of extra tissue and fracture point trauma. After the fracture is healed, all implants can be eliminated from patient body. Biodegradable polymers used for bone scaffold are propylene fumarate and chitosan, with a copolymer such as lactide glycolide. Khan et al. (Ambrosio, Sahota, Khan, & Laurencin, 2001) prepared calcium phosphate scaffold. This material



CHAPTER 2 Foundation of composites

was inoculated for 8 weeks in simulated body fluid. Simulated body fluid pH and scaffold mass decreased firstand than increased. The composite scaffolds increased reprecipitation of calcium phosphate simulated body fluid for the implant. Researchers are paying more attention to the bioactive ceramics because of their excellent bone tissue binding nature. Reproduction of the osteoblasts occurs due to contact between blood plasma and HA layer coating on the surface of embedded composite. In most of studies HA particles are reinforced in highdensity polyethylene (HDPE) matrix as a bone replacement material (Bonfield, Wang, & Tanner, 1998; Di Silvio, Dalby, & Bonfield, 2002; Ladizesky, Pirhonen, Appleyard, Ward, & Bonfield, 1998; Salernitano & Migliaresi, 2003; Wang & Bonfield, 2001; Wang, Deb, & Bonfield, 2000; Wang, Joseph, & Bonfield, 1998). The successful marketing of HA-based composite was started in 1995 by a company name HAPEX. The bone substitutes manufactured and traded by HAPEX suffered from drawbacks of strain failure, low stiffness and strength as compared with cortical bone. This is why these substitutes cannot be used in load-bearing applications.

2.9.2 Orthopedic As summarized by Evans and Gregson (1998) composite materials are widely used in orthopedic applications, especially in bone fixation plates, hip prosthesis, bone cements, and bone grafts. The most common materials for hip joint supplement of femoral stem are 3161 stainless steel and some alloy of cobalt, chromium and titanium. These alloys are 10 times than the bones in body they are replacing. Cortical bone in the body has stiffness of 16 GPa and tensile strength of 92 MPa (Katz, 1966), whereas titanium has tensile strength and stiffness of approximately 800 MPa and 110 GPa, respectively, which is clearly more as compared to the bone it replaces. This causes unpleasant bone reshaping and stress shielding, resulting in a loss of bone mass and implant displacement over time, particularly in the proximal part. Fiber reinforcements can be used to match tensile strength of bone. Carbon fibers reinforced in polysulfone matrix have stiffness and tensile strength of about 170 GPa and 900 MPa, respectively. Carbon fiber polysulfone composites are difficult to produce and have low durability but they continue to be fabricated for the inherent advantages of flexibility and radiolucency. Carbon debris from the composites can be mitigated by polishing with titanium alloy and HA (Baˇca´kova´, Stary´, Kofroˇnova´, & Lisa´, 2001). A resorption-rich bone plate is required to eliminate the need for another implant after complete fracture fixation healing. The rate of degradation must be controlled and degradation by-products must be nontoxic that aids to balance the mechanical parameters such as stiffness loss in implantation and increases healing strength. Composite bone plates of carbon fiber with PLA and calcium phosphate fiber with PLA are both fully resorbable (Iftekhar, 2004). These composites however do not have suitable mechanical properties and degrade rapidly. Bone cement is used to fill the voids and enhance adhesion between host bone tissues

2.9 Applications of biocomposites

and implant bone which has been used to reinforce with fiber matrix to prevent loosening and enhance strength. PMMA powder mixed with methacrylate-type monomer is used as typical bone cement during fixation. Creep deformation and fatigue life of PMMA matrices can be enhanced by adding a low volume fixation of graphite, carbon, and Kelvar fibers (Kelly, Cahn, & Bever, 1994).

2.9.3 Dental Composites have so far been the most successful in the dental field. There are rigorous design requirements, which are hard or difficult to obtain from materials such as ceramic alloys and metal alloys. Whether it is to fabricate crowns, restore fillings, or fill in missing teeth, the composite must matched the color and diaphanous with adjacent teeth and keep its shine (Furtos, Silaghi-Dumitrescu, Lewandowska, Sionkowska, & Pascuta, 2016). The antiwear stress and fracture must be matched with the strength of above tooth. The tooth must be isotropically stable inside the mouth and withstand the varying temperatures of foods. Organic and inorganic inclusions added to polymer resin matrix are used in repairing fillings as shown in (Fig. 2.5). Ceramics, calcium silicate, calcium fluoride, and crystalline quartz are some of the particles used to increase strength and wear resistance. Fatigue fractures and water absorption can be reduced by using silica particles as reinforments and treating the composites with a silane coating. These treatments may improve retention of matrix (Beatty, Swartz, Moore, Phillips, & Roberts, 1998). Normally, the size of the filler particles are between 20 nm and 50 nm in size. Filler volume of about 80% can be used to fabricate composites. Fused silica particles of between 20 and 50 nm in size ared used in microfilled dental resin to a volume of 42%. These composites are colorless and can be coated with an opaque gloss however, the mechanical strength of these composites is not enough for posterior teeth and hard to handle the coating due to low viscosity of gloss (Furtos et al., 2016). Particle size from 0.1 to 10 μm are used in hybrid dental resin. These dental resins provide easy handling due to higher filler volume around 80% particle content and higher viscosity. Hybrid resins have low water absorption compared with microfilled resin. Commercial dental resin could change shape due to polymerization shrinkage of 1.2% 2.9% and up to 1.5% water absorption (Beatty et al., 1998). These composites have difficulty adhering to dentine, causing fitting and leakage problems. Using all ceramic dental composites can enhance stress bearing on crowns and bridges as shown in (Fig. 2.5). Decreasing fractures in is one of the very important challenges for dental composites. A common type of composite is In-Ceram made from alumina glass, in which there are slip casting molds, sintering of a skeleton of alumna particle, followed by melt seepage of glass into the porious areas (Wolf, Vaidya, & Francis, 1996). Fracture toughness was not affected by thermal expansion mismatch between alumina and glass. It is necessary to match the hardness of composites with the enamel of opposing teeth to reduce the wear stress. Hardness of teeth can be enhanced by coating composites with alumina silicate calcium phosphate.



CHAPTER 2 Foundation of composites

FIGURE 2.5 (A) Natural tooth crown with screw at lower part along with natural tooth and (B) broken tooth image before tooth filling and (C) after the filling of tooth with composite.

Posterior and anterior teeth can be replaced by composite materials. The broken teeth replaced by the oral prostheses done by PMMA provides excellent mechanical properties to the posterior teeth. The composites in the dentistry field has several crucial requirements, such as long-term durability, dimensional stability, wear resistance, minimal shrinkage in polymerization, and higher strength. Filling microcavities that may form are responsible for the excellent mechanical and wear resistance properties. The adhesion between the filler and microcavities results from lack of material involvement (Ensaff, O’Doherty, & Jacobsen, 2001).

2.9 Applications of biocomposites

Orthodontic archwires are another application of dental composites. Fiber volume fraction ranges from 30% to 75%, depending on the glass fiber yarns used the strength and the modulus of titanium wires. Hyoxyapatite ceramic particles reinforced in polyethylene matrix produce orthodontic brackets, which have good isotropic properties and excellent adhesion to enamel.

2.9.4 External prosthetic and orthotics Skeletal bone is an important part of human body that supports and protects the essential organs. Red blood cells and white blood cells are produced by the bones. Bone grafting is an invasive procedure that transplants bone into the injured bone in order to heal a fracture that is tremendously complex. Bone grafts serve as a structural framework for limb formation, maturation, and remolding. Several materials are used as bone repairing materials as shown in Fig. 2.6. Wood, aluminum, and leather are traditional orthotics and prosthetics materials and these are being widely substituted with extraordinary composites and thermoplastics. Fiberreinforced composites are light weight, can be very small in size, and are safe to use; that is why they have become a very attractive option in this area. These traditional composites are designed and manufactured using a specific thermoset to be very strong and exterior to the body. Composites have been used to interface transtibial (TT) and transfemoral prostheses (TP) with the residual limb as well as to spread out the load over the surface (Paul, 1999). Only 1 2 kg weight can be targeted by TT and TP prosthesis, which makes carbon-reinforced composites ideal. Socket frame is made up of tape of carbon fiber and resin. Stiffness of bone socket and grafts can be tailored using methyl methacrylate, which is a blend of rigid and flexible polymeric matrix.

FIGURE 2.6 Different approaches for bone repair.



CHAPTER 2 Foundation of composites

Stainless steel in artificial arms has been replaced by carbon fiber-reinforced (CFR) tubes. Carbon fiber/nylon hybrid composites have exhibited excellent contact resistance as compared to single-carbon nylon composites. Injection molding technique is used to fabricate CFR/nylon 6, 6 hybrid composites. These fabricated composites have excellent vibration damping and shock absorption properties. For the prosthetic foot unit (shown in Fig. 2.6), various composites have the property to store flexural energy and reuse it during motion of body. According to individual patient needs and their characteristics, modification of the foot unit and prosthesis have been done to preserve the flexural energy of the implant. For this requirement the flex foot unit contains long CFR epoxy composites rod that preserves the unbroken length of prosthesis (Fu et al., 2015). In the carbon-copy foot, posterior Kevlar-reinforced nylon block and anterior CFR plastic leaf springs were the materials that are used to produce keel and provide dual stage support to flexion (Mir et al., 2018). At last, nylon-reinforced silicon elastomers have been used to construct a tough plaster to make a bendable foam that is wrapped over the simulated body part. The orthotics were originally designed to support tissue injury. These days their application can be found in splinting material for cast formation, replacing the old cotton and plaster of Paris. Besides strength, they have the advantages of better X-ray transmission and lower water adsorption. Fig. 2.7 depicts different types of implanting devices in human body.

FIGURE 2.7 Different types of prosthesis and replacement in body.

2.9 Applications of biocomposites

2.9.5 Biocompatibility on skin Skin is considered as the first organ of the immune system as it fights against the harmful antigens and pathogens. Skin can be damaged in several ways, such as burns, cuts, injuries, infections, etc., but cannot heal itself. A critical requirement in the field of tissue engineering, is to develop biocompatible and biodegradable material for skin healing and repairing (Jeong, Park, & Lee, 2017; Zhao et al., 2019). The structure of the grafts should match the structure of natural skin extracellular matrix. Several natural and synthetic hydrogels are being fabricated and developed for skin regeneration such as collagen, hyaluronic acid, polyviny alcohol, and polyethylene glycol (Bahadoran, Shamloo, & Nokoorani, 2020). Thin films made of polyvinyl alcohol and sodium alginate were synthesized for skin burns (Kamel, Abd El-messieh, & Saleh, 2017). The wound healing properties of chitosan can be more efficient by reinforcing it with banana peel powder. Kamel and his colleagues synthesized a nanocomposite membrane from banana peel powder. Banana peel powder acts as an ionic cross-linker (Ghiasi, Chen, Rodriguez, Vaziri, & Nazarian, 2019). Nanocomposite membrane has high surface-to-volume ratio which enhances cell proliferation and cell migration. Another compound for wound dressing is spider web and silk. Spider’s web can stop the bleeding and heal the wound rapidly.

2.9.6 Healing of fracture and wound dressing A complex process that involves unique and highly integrated series of events is used to heal fractures in the body. The process of repairing a endochrondal fracture begins with pro-inflammatory and ends with remolding phase (Neumann & Epple, 2006). In initial stage a hematoma is formed from the blood vessels ruptured by the injury. Inflammatory cells invade hematoma and remove the necrotic debris. A combination of degranulating macrophages, leukocytes, platelets, and mast cells penetrates the fractured area and amplifies the initial inflammatory responses by activating additional pro-inflammatory cytokines and peptide signaling molecules that trigger and promote growth and repair, as well as begin to clean up necrotic debris. In a typical trauma, the fracture undergoes several types of tissue malfunction. Naturally using bone grafts can make the situation even worse, therefore there is high demand for bone cement as a substitute. Unfortunately medical application of xenograft is generally carriers’ viral infection. Xenograft is generally use for transplantation of tissue, organ of one other species to human body which unfortunately causes viral infection. Other disadvantage of xenograft is it is easy resorption and low osteogenicity as compare to autogenous bone in body. Calcium phosphate is widely used as a bone substitute material due to their chemical similarity to the mineral component of mammalian bone and teeth (Gazdag, Lane, Glaser, & Forster, 1995). Some commonly used calcium phosphate cements are listed in Table 2.1. Most importantly, it is nontoxic and biocompatible.


Table 2.1 Existing calcium phosphate and its properties. Molar ratio of Ca/P 0.5 1.0 1.33 1.5

Material name

Chemical formula

Solubility at 25 C, -log(Ks)

Solubility at 25 C, (g/L)

Range of pH stability in aqueous solutions at 25 C

Mono-calcium phosphate monohydrate (MCPM) Dicalcium phosphate dihydrate (DCPD), Octacalcium phosphate (OCP) β-Tricalcium phosphate (β-TCP)



Approx. 18

Between 0.0 and 2.0



Approx. 0.088

Between 2.0 and 6.0



Approx. 0.0081

Between 5.5 and 7.0



Approx. 0.0005

Stable at temp 100 C

2.9 Applications of biocomposites

Calcium phosphate cement has exhibited bioactive behavior and ability to integrate into living tissue by the same process active in remodeling healthy bone. Calcium phosphate cement is used to fill the defect after the injury or breakage of bone (Fig. 2.8). The major drawback of calcium phosphate cement is its poor mechanical properties resulting in poor load-bearing capacity. For a better result calcium phosphate cement is reinforced with a different material of same or more biocompatibility and having more strength. Many patients suffer from blood loss and infection during and after a personal injury. Slow wound healing generates microbial infections due to its molecular and cellular behavior that can be controlled by growth of secreting cells. The fundamental wound healing process is fabrication of collagen fibers due to transportation of fibroblast to the wound. Wound and its nearby location is safeguard by wound dressing. Some of wound dressing materials are polymers, cotton, hydrofibers, films, foams, and alginates. Some important examples of naturally extracted wound dressing materials are silk sericin and spiders’ web derived from silkworm and spiders respectively, due to their antibacterial, moisture absorption, and oxidation resistance properties. These materials immediately stop the blood loss and healing of the wound can begin. Some synthetic wound dressing materials are kaolin/polyurethane, silver-cobaltdoped bioactive glass nanoparticles, cross-linked polymer network films, etc. (Zhang, 2002).

FIGURE 2.8 Radiographs demonstrating the use of bio-net materials. (A) A posterior image of the both leg shows the use of calcium phosphate bone cement and additional Fixation device made of u-HA reinforced polymer (arrows) to fill the defect after bone brake. Bone cement can be used as an adjunct to increase the purchase of screws during fracture fixation (arrow). (B) Image (1) showing before treatment and image (2) showing after treatment, a biodegradable polymer impart.



CHAPTER 2 Foundation of composites

2.10 Fabrication techniques of biomedical composites There are several approaches for fabrication of the constituents of biomedical composites Several of the approaches are taken from a different industry, but some methods are developed to satisfy a particular design or fabrication challenge. Selection of the fabrication technique depends up the requirements of the particular part, such as the design, application and the material to be used. Molds are often used to provide the required during composite fabrication process. Specialized tools are required to ensure the correct shape during the molding and the curing process.

2.10.1 Hand layup molding Hand layup is the most common fabrication approach for polymer-based thermoset composites. Hand layup technique comprises arranging plies (sheets of fiber) by hand onto an implement use for making coated stack. The stack is placed onto the mold, if being used and they system placed under a vacuum. Resin is then infused into the plies of fiber. There are a number of curing techniques but the most basic is simply to permit the composite to cure at a particular temperature. The whole process of curing is performed in vacuum. Application of heat can accelerate curing. High heat and constant pressure are required for curing high-performance thermoset polymer-based composites. Autoclaves can also be used for curing, but are expensive to purchase and to operate. Many parts can be cured simultaneously by manufacturers having autoclaves. Autoclave temperature, vacuum, pressure, and inert atmosphere are controlled by the computer systems. The computer system also provides remote supervision of curing process. Thin laminates are cured by methods using an electron beam (E-beam). This is an effective method. In Ebeam curing, the amalgamated layup is exposed to a stream of electrons that delivers radiation causing polymerization and cross-linking in sensitive resins. An analogous manner is curing through X-ray radiation or microwave. In thermoset resin a photoinitiator has been added, and this initiator is activated by ultraviolet (UV) radiation. Activating the photoinitiator in the resin sets off a cross-linking reaction. UV radiation curing is termed a fourth alternative for curing. Only light permeable resin and reinforcement can be cured using this method.

2.10.2 Open contact molding method One-sided molds can be inexpensiveand the basic method for creating fiberglass composites in one-side molds is the open contact molding method. This is lowcost and effective method to fabricate composites. Open molding technique is typically used for cabs of truck, hulls of boat, decks, bathtubs, shower stalls, and other large simple shapes. In this process a rigid one-sided mold is used to provide the surface finish to one side of a component. The thickness of composite depends on how much of composite piles are placed in the mold. The plies can

2.10 Fabrication techniques of biomedical composites

be placed in the mold either by hand layup or by spray up. To prepare mold surface, a gel coating is applied on the inside of the mold to help with the release step. The sprayed gel is then cured and mold is ready. In the spray up process, reinforcement fibers are sprayed into a mold using a chopper gun, which slices the fibers into short lengths. These chopped fibers are then sprayed directly onto a stream of resin in order to make the resin and fibers adhere to each other perfectly. In this process the volatile organic compounds reduced by activated piston that further activates the spray guns and nonfluid impingement spray heads distribute gel coating on fiber and after gel coating the resin is cured in low pressure along with fiber/particulate. Workers compressed the laminate with a roller in next step of the method. Other core material such as wood, foam, etc. may then be added and the core may then be sprayed with another layer. The molded portion is then cooled, cured, and taken out from mold. Hand layup or spray up technique require fewer number of labors. Faster production rate of resin infusion process makes the industries to switch to hand layup technique as an alternative process for fabrication and encourage the fabricators to process this method wherever possible.

2.10.3 Liquid molding and injection molding Resin transfer molding (RTM) is a common alternative for fabrication and is also known as liquid molding. The RTM process has very impressive results and benefits. Generally, the materials used in RTM are less costly and can be stored at room temperature as compared to pre-impregnated (prepreg) materials. This method generally produces a very thick shape and eliminates most postfabrication work. All exposed surface gets smooth finish and complex parts are dimensionally accurate having good surface details with this method. Before closing the mold, it is possible to put inserts inside the preform. The RTM process allows for integration of other hardware and additional core materials. RTM process requires less cycle time to complete and could be automated for greater effieiency. It reduces a cycle time from several days to just hours or perhaps minutes. In RTM process before injection into mold, the resin and catalyst are mixed, where in reaction injection molding (RIM) two separate streams, one of resin and one of the catalyst, are injected into the mold. Therefore all the chemical reactions take place inside mold rather than in the dispensing head. In the automobile industry, RIM and other quick reaction approaches are combining together fabricate structural parts but without A-class finish. The fiber and resin mixture can be sprayed up onto a vacuum mold by means of programmable robots. Robotic spray up can be directed to regulate fiber alignment.

2.10.4 Vacuum resin transfer molding process Vacuum resin assisted transfer molding (VARTM) is a fast emerging molding technique. The difference between resin transfer molding process and vacuum-



CHAPTER 2 Foundation of composites

assisted process is the vacuum. VARTM can operate at moderate heat and does not require heavy, expensive equipment. VARTM can produce large complex parts inexpensively at one time. As the whole process in VARTM is carried out in presence of vacuum tight seal; therefore a canopy/plastic film is placed at the highest and it is a two-sided mold, where resin is injected from one side and reinforcement injected from another side. The resin enters the mold through various ports and feed lines, termed manifold ports. These are drawn by the vacuum that facilitates the wetting of fibers in matrix. There are many applications of structures made from theis method, such as bus, car, bikes, marine transportation, and construction work on ground, many infrastructural structures in building, medical field, etc. This method is first used by Boeing cooperation (Chicago III) and by NASA. The first firm that used this process was small and provided laminate pieces for aerospace without the use of an autoclave.

2.10.5 Compression molding Compression molding is a high-volume and high-pressure molding process appropriate for producing strong pieces. This method employs durable metal dies which are very expensive. Thermoplastic composite can be molded with fibers, fabrics, optical strands, etc. When production quantity requirements exceeds 15,000 parts, use of the compression molding technique is very beneficial. Compression molding technique produces very simple structures, has lower tooling costs and less waste. as compared to other techniques In this method the molding material is preheated in an oven before being added to the molding cavity. A large hydraulic press is required to compress the top of the mold onto the material inside the mold to form the desired shape. The material is then cured by means of pressure. By using sheet molding compound (SCM), large parts can be fabricated in forged mold dies kept in preheated oven. SCM is a sheet of composite fabricated by inserting fiberglass or other fiber strands in chopped form and using glue or resin paste to combine two or more sheets. Low-pressure SCM offers a low capital investment free from volatile organic compounds emission and potentially a supreme quality surface finish to the structure part. Automotive industry suppliers use carbon fiber-reinforced sheet molding material, to take advantage of the benefits of carbon, its high strength and stiffness. During the oven cure, the discharge gases of inserted material are trapped within the microcracks of cured compound. These microcracks can be prevented using SMC technique. Composite producers in industrial markets are formulating their own resins and compounding SMC inhouse to satisfy need for customized material fabrication, in applications that require UV radiation protection, impact and moisture resistance, or have high surface-quality demands. Injection molding can be a fast, high-volume, lowpressure, and closed process using, most typically, reinforced thermoplastics, such as nylon reinforced with chopped glass fiber. In last 20 years automated injection molding process has seized some market operated by thermoplastic and metal casting manufactures. Transfer molding is a closed mold process where a nozzle

2.10 Fabrication techniques of biomedical composites

forces the material into the molder cavity where the material is settled under the application of temperature and pressure. Filament binding is a repeatable method that has low costing of material. Structural part yields by filament method have high strength. Golf shafts are made by using filament winding method. Pressure vessels, rods, pipe, tubes, etc. are used in several other businesses. Glass fibers and resins have been used for many years such as RTM in pultrusion process, but in last 10 years this method has set roots in advance composite applications. During this low-cost, simple, and continuous process, a forged resin bath is required to heat the resin so that reinforced fibers can pull through it to make specific shapes by passing through many forming bushings. The shape and curing of fabric takes when the fabric moves through the heated die filled with resin. After cooling, desired length of the fabric and resin mixture is formed. Requirement of post processing is negligible to provide smooth finish to the structures by converting the fibers and resin into thermoplastic polymer. Pultrusion of good range of continuous, solid or hollow profiles takes place and therefore the process is often custom-tailored to fit specific applications.

2.10.6 Tube rolling Tube rolling may be a long-standing composite manufacturing process which will produce finite-length tubes and rods. It is mainly applicable to small-diameter cylinder having diameter of approximately 6 in. or 152 mm that will be rolled properly. Typically an adhesive prepreg fabric is employed depending on the part. The fabric is precut in shapes that are styled to attain the essential ply schedule for the applications. A cylindrical tube is rolled over the surface under certain pressure to compress the other material pieces and to remove the part of the bulk from the flat surface. Only primary rows of fibers fall on verity 0-degree axis by rolling mandrel.

2.10.7 Automated fiber/tape placement process Fibers can be continuously placed under a mandrel at high speeds using a programmable robotic head to cut and then clamp the tows. More than 32 tows can be clamped and cut simultaneously. The mandrel cuts the fibers into a shortest possible length. The automated machine attached to dual mandrel tubes or station produces fiber more efficiently and increases production. A 5-axis overhead bridge-like structure supports the fiber placement head and is fitted to a winder of filament to deliver custom system. Automated fiber placement (AFP) has certain advantages such as less processing speed, material scrap, wastage, labor cost, and improved part uniformity. Often the method is employed to provide complex shapes having bulky parts. Automated tape laying (ATL) is a similar, faster automated process within which prepreg tape, instead of single tows, is used to make structure. It is used for fragments having complicated angles. Tape layup is flexible, allowing breaks within the process and straightforward alignment changes,



CHAPTER 2 Foundation of composites

and it may be adapted for both thermoset and thermoplastic materials. The top of the machine contains a coil of tacky winder and a tape cutter. In either case, the pinnacle could also be set at the tip of a multiple axis robot that rotates around a cylindrical tube. ATL is quicker than AFP and might place more material over wider distances; AFP is well matched to shorter courses and places the material over the counter surfaces.

2.11 Conclusion The composite materials have superior properties for application in medical field, which makes them a promising material in research and development. Biomedical composites are cost effective, provide better strength and stiffness to the parts they are replacing, composed of natural, biodegradable, and biocompatible matrices with certain reinforcements. Open molding, injection molding, and compression molding are some highly reported techniques of fabrication of composites. Polymer composites have received a great deal of attention from researcher and medical professional due to their great effectiveness in dental technology, bone cement technology, bone fracture healing, tissue engineering, prosthesis, cancer treatment, etc. Polymer-based bone scaffolds show great strength, biodegradability, biocompatibility, and high cell adhesion, and low inflammatory reaction upon implantation. However, composites have certain limitations, such as having poor cell attachments and releasing acidic by-products. These drawbacks could be assuaged in future by integrating nanobioreinforcements, with more biocompatibility, having bioactive molecules and nontoxic nature.

References Ambrosio, A. M., Sahota, J. S., Khan, Y., & Laurencin, C. T. (2001). A novel amorphous calcium phosphate polymer ceramic for bone repair: I. Synthesis and characterization. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 58(3), 295 301. Bahadoran, M., Shamloo, A., & Nokoorani, Y. D. (2020). Development of a polyvinyl alcohol/sodium alginate hydrogel-based scaffold incorporating bFGF-encapsulated microspheres for accelerated wound healing. Scientific Reports, 10(1), 1 18. Baˇca´kova´, L., Stary´, V., Kofroˇnova´, O., & Lisa´, V. (2001). Polishing and coating carbon fiber-reinforced carbon composites with a carbon-titanium layer enhances adhesion and growth of osteoblast-like MG63 cells and vascular smooth muscle cells in vitro. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials, 54(4), 567 578. Beatty, M. W., Swartz, M. L., Moore, B. K., Phillips, R. W., & Roberts, T. A. (1998). Effect of micro filler fraction and silane treatment on resin composite properties.


Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and the Australian Society for Biomaterials, 40(1), 12 23. Bharath, K. N., & Basavarajappa, S. (2016). Applications of biocomposite materials based on natural fibers from renewable resources: A review. Science and Engineering of Composite Materials, 23(2), 123 133. Bonfield, W., Wang, M., & Tanner, K. E. (1998). Interfaces in analogue biomaterials. Acta Materialia, 46(7), 2509 2518. Di Silvio, L., Dalby, M. J., & Bonfield, W. (2002). Osteoblast behaviour on HA/PE composite surfaces with different HA volumes. Biomaterials, 23(1), 101 107. Ensaff, H., O’Doherty, D. M., & Jacobsen, P. H. (2001). The influence of the restoration tooth interface in light cured composite restorations: A finite element analysis. Biomaterials, 22(23), 3097 3103. Evans, S. L., & Gregson, P. J. (1998). Composite technology in load-bearing orthopaedic implants. Biomaterials, 19(15), 1329 1342. Furtos, G., Silaghi-Dumitrescu, L., Lewandowska, K., Sionkowska, A., & Pascuta, P. (2016). Biocomposites for orthopedic and dental application. Key Engineering Materials, 672. Fu, S., Yu, B., Duan, L., Bai, H., Chen, F., Wang, K., . . . Fu, Q. (2015). Combined effect of interfacial strength and fiber orientation on mechanical performance of short Kevlar fiber reinforced olefin block copolymer. Composites Science and Technology, 108, 23 31. Gazdag, A. R., Lane, J. M., Glaser, D., & Forster, R. A. (1995). Alternatives to autogenous bone graft: efficacy and indications. JAAOS-Journal of the American Academy of Orthopaedic Surgeons, 3(1), 1 8. Ge, S., Kang, X., & Zhao, Y. (2011). One-year biodegradation study of UHMWPE as artificial joint materials: Variation of chemical structure and effect on friction and wear behavior. Wear, 271(9 10), 2354 2363. Ghiasi, M. S., Chen, J. E., Rodriguez, E. K., Vaziri, A., & Nazarian, A. (2019). Computational modeling of human bone fracture healing affected by different conditions of initial healing stage. BMC Musculoskeletal Disorders, 20(1), 1 14. Giurgiutiu, V. (2015). Structural health monitoring of aerospace composites. Iftekhar, A. (2004). Biomedical composites. Standard handbook of biomedical engineering and design. New York: McGraw-Hill. Jeong, K.-H., Park, D., & Lee, Y.-C. (2017). Polymer-based hydrogel scaffolds for skin tissue engineering applications: A mini-review. Journal of Polymer Research, 24. Kamel, N. A., Abd El-messieh, S. L., & Saleh, N. M. (2017). Chitosan/banana peel powder nanocomposites for wound dressing application: Preparation and characterization. Materials Science and Engineering: C, 72, 543 550. Katz, J. (1966). `ıOrthopedic applications, ıˆ in Biomaterials Science, BD Ratner. Kelly, A., Cahn, R. W., & Bever, M. B. (1994). Concise encyclopedia of composite materials. Revised Edition. New York: Pergamon Press. Ladizesky, N. H., Pirhonen, E. M., Appleyard, D. B., Ward, I. M., & Bonfield, W. (1998). Fibre reinforcement of ceramic/polymer composites for a major load-bearing bone substitute material. Composites Science and Technology, 58(3 4), 419 434. Lau, K. T., Ho, M. P., Au-Yeung, C. T., & Cheung, H. Y. (2010). Biocomposites: Their multifunctionality. International Journal of Smart and Nano Materials, 1(1), 13 27.



CHAPTER 2 Foundation of composites

Malhotra, S. K., Goda, K., & Sreekala, M. S. (2012). Part one introduction to polymer composites. Polymer Composites, 1, 1 2. Mir, M., Ali, M. N., Barakullah, A., Gulzar, A., Arshad, M., Fatima, S., . . . Asad, M. (2018). Synthetic polymeric biomaterials for wound healing: A review. Progress in Biomaterials, 7(1), 1 21. Neumann, M., & Epple, M. (2006). Composites of calcium phosphate and polymers as bone substitution materials. European Journal of Trauma, 32(2), 125 131. Pal, H., Jit, N., Tyagi, A. K., & Sidhu, S. (2011). Metal casting A general review. Advances in Applied Science Research, 2(5), 360 371. Paul, J. P. (1999). Strength requirements for internal and external prostheses. Journal of Biomechanics, 32(4), 381 393. Pye, A. D., Lockhart, D. E. A., Dawson, M. P., Murray, C. A., & Smith, A. J. (2009). A review of dental implants and infection. Journal of Hospital Infection, 72(2), 104 110. Ramakrishna, S., Mayer, J., Wintermantel, E., & Leong, K. W. (2001). Biomedical applications of polymer-composite materials: A review. Composites Science and Technology, 61(9), 1189 1224. Salernitano, E., & Migliaresi, C. (2003). Composite materials for biomedical applications: A review. Journal of Applied Biomaterials and Biomechanics, 1(1), 3 18. Scala, E. P. (1996). A brief history of composites in the US-The dream and the success. JOM, 48(2), 45 48. Seal, B. L., Otero, T. C., & Panitch, A. (2001). Polymeric biomaterials for tissue and organ regeneration. Materials Science and Engineering: R: Reports, 34(4 5), 147 230. Selzer, R., & Friedrich, K. (1997). Mechanical properties and failure behaviour of carbon fibre-reinforced polymer composites under the influence of moisture. Composites Part A: Applied Science and Manufacturing, 28(6), 595 604. Sharma, P., Bhanot, V. K., Singh, D., Undal, H. S., & Sharma, M. (2013). Research work on fiber glass wool reinforced and epoxy matrix composite material. International Journal of Mechanical Engineering and Robotics Research, 2, 106 124. Wang, M., & Bonfield, W. (2001). Chemically coupled hydroxyapatite polyethylene composites: structure and properties. Biomaterials, 22(11), 1311 1320. Wang, M., Deb, S., & Bonfield, W. (2000). Chemically coupled hydroxyapatitepolyethylene composites: processing and characterisation. Materials Letters, 44(2), 119 124. Wang, M., Joseph, R., & Bonfield, W. (1998). Hydroxyapatite-polyethylene composites for bone substitution: effects of ceramic particle size and morphology. Biomaterials, 19 (24), 2357 2366. Wolf, W. D., Vaidya, K. J., & Francis, L. F. (1996). Mechanical properties and failure analysis of alumina-glass dental composites. Journal of the American Ceramic Society, 79(7), 1769 1776. Zhang, Y. Q. (2002). Applications of natural silk protein sericin in biomaterials. Biotechnology Advances, 20(2), 91 100. Zhao, Y., Li, Z., Song, S., Yang, K., Liu, H., Yang, Z., . . . Lin, Q. (2019). Skin-inspired antibacterial conductive hydrogels for epidermal sensors and diabetic foot wound dressings. Advanced Functional Materials, 29(31), 1901474.


Biopolymer-based composites for drug delivery applications—a scientometric analysis


Kunal Pal1, Deepti Bharti1, Preetam Sarkar2 and Doman Kim3 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 3 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

3.1 Introduction Drug delivery vehicles are regarded as the formulations or devices that are responsible for delivering drugs. Controlled drug delivery systems are a specific type of formulations and devices that allow controlling the rate of release of the drugs to maintain their level at the site of action. Such systems can also be designed to deliver drugs at a specific time or as per the demand. These systems form an interface between the drug and the patient. Biopolymers (e.g., chitosan, alginate, hyaluronic acid, etc.) have been explored for designing controlled drug delivery systems. These are the polymers that are available in nature in abundance. Fig. 3.1 (Balart, GarciaGarcia, Fombuena, Quiles-Carrillo, & Arrieta, 2021) summarizes the classification and sources of the biopolymers. Due to this reason, they are inexpensive. The biopolymers are inherently biocompatible and nonimmunogenic (Gheorghita, AnchidinNorocel, Filip, Dimian, & Covasa, 2021). They are available in a wide range of chemistries, which allow researchers to design delivery systems with desirable properties. It is possible to deliver drugs at a specific site. For example, it is possible to protect some medications from the harsh gastric pH and deliver the same to the duodenal or intestinal region (Zhao, Maniglio, Chen, Chen, & Migliaresi, 2016). On the contrary, some drugs are to be released in the gastric region of the gastrointestinal tract. Similarly, some of the biopolymeric formulations can cross the blood brain barrier to deliver drugs to the central nervous system. Further, the chemical modifications (e.g., carboxylation, thiolation, acetylation, and conjugation) of the biopolymers can be carried out (Dmour & Taha, 2018). The modifications can expand the functionality and applications of the biopolymers. For example, PEGylation of the Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 3 Biopolymer-based composites

FIGURE 3.1 Classification and sources of biopolymers. Reproduced from Balart, R., Garcia-Garcia, D., Fombuena, V., Quiles-Carrillo, L., & Arrieta, M. P. (2021). Biopolymers from natural resources. Polymers, 13(15). under Creative Commons License.

biopolymeric nanoparticles allows the researchers to develop stealth delivery systems that can have an increased residence time in the blood (Suk, Xu, Kim, Hanes, & Ensign, 2016). This, in turn, improves the bioavailability of the drugs and their efficiency. The properties of the biopolymer-based systems may further be improved by developing their composites. These types of formulations may provide improved therapeutic benefits over the conventional delivery systems. In this chapter, we hereby perform the scientometric analysis to analyze how the use of biopolymeric composites in drug delivery systems has evolved over the years. The collection of data was carried out from the Web of Science (WoS) database. Analysis of the data was carried out using the VoSviewer software, a freeware. After analyzing the data, we identify the commonly used biopolymers for developing composite-based drug delivery systems. Subsequently, we discuss the properties of these biopolymers. Finally, we recognize the most-cited literature in the said field and briefly discuss the research finding in that literature.

3.2 Scientometric analysis The articles for the scientometric analysis were searched using the keywords: biopolymer (All Fields) and composite (All Fields), and “drug delivery”

3.2 Scientometric analysis

(All Fields). The search was carried out in the WoS database on October 10, 2021. The search returned with 451 publications. Among the publications, there were 313 articles, 137 review articles, seven proceeding publications, six early access publications, one book chapter, and one meeting abstract. Further 450 publications were in the English language, while one publication was in the Chinese language. Subsequently, the publications in the English language and categorized as articles were selected for further analysis. The application of these filters returned with 312 publications. It was observed from the results that the first publication on the proposed topic was during the year 2002. The analysis of the data deciphered that 74 articles were published as Open Access articles. Fig. 3.2 deciphers the growth of the publications over the years till the current day. It is important to note that the biopolymeric composite based drug delivery systems have evolved considerably in the last decade. Since the year 2016, there have been more than 30 publications each year. The highest number of publications was achieved in the year 2020. Fig. 3.3 shows the TreeMap chart of the top five publishers who have published the articles. It could be seen that the highest number of publications were published by Elsevier (134 articles) followed by Wiley (49 articles), Springer Nature (22 articles), MDPI (22 articles), and Royal Society of Chemistry (17 articles), respectively. The top five journals that published the publications were Journal of Applied Polymer Science (23 articles), International Journal of Biological Macromolecules (21 articles), Carbohydrate Polymers (14 articles), Materials Science Engineering C: Materials for Biological Applications

FIGURE 3.2 Histogram of publications on a time scale.



CHAPTER 3 Biopolymer-based composites

FIGURE 3.3 Most number of articles published by the publishers.

(13 articles), and Journal of Drug Delivery Science and Technology (7 articles), respectively. It was interesting to note that all the articles were from the Elsevier Publishing group. Most articles were published from the countries People’s Republic of China (52 articles), India (48 articles), Iran (37 articles), the United States (37 articles), and Spain (22 articles), respectively. Thereafter the bibliometric records of the articles were downloaded as the Tab delimited file. The data so obtained were then analyzed using Vosviewer software. The Vosviewer software is a free software that several researchers are using to analyze bibliometric data.

3.2.1 Coauthorship analysis Initially, coauthorship analysis was carried out. The details of the coauthorship analysis when the analysis was carried out using the relationship of the authors, who had at least three articles, are tabulated in Table 3.1. It can be observed that the total link strength, which provides information regarding the collaborative research amongst the authors, was highest for Giuseppe Cavallaro, Giuseppe Lazzara, Lorenzo Lisuzzo, and Stefana Milioto. These authors also shared an equal number of documents and citations, which indicated that these authors were collaborating. Fig. 3.4 summarizes the coauthorship analysis among the authors. The analysis of Fig. 3.4 suggests that the authors Giuseppe Cavallaro, Giuseppe Lazzara, Lorenzo Lisuzzo, and Stefana Milioto are collaborators. This group formed the largest network. It can also be observed from Fig. 3.4 that the authors Hriday Bera and Aldo R. Boccaccini are also collaborators. However, the number of documents and the citations of these authors were different (Table 3.1). This is suggestive of the fact that the author Hriday Bera, who has four articles against

3.2 Scientometric analysis

Table 3.1 Coauthorship analysis at the individual level of the authors. #




Total link strength

1 2 3 4 5 6 7 8 9 10

Cavallaro, Giuseppe Lazzara, Giuseppe Lisuzzo, Lorenzo Milioto, Stefana Abbasi, Yasir Faraz Bera, Hriday Boccaccini, Aldo R. Castro, Guillermo R. Holban, Alina Maria Tan, Huaping

3 3 3 3 3 4 3 3 3 3

100 100 100 100 12 44 150 101 17 15

9 9 9 9 3 3 0 0 0 0

FIGURE 3.4 Author network analysis.

his name, has also worked in the aforesaid field wherein Aldo R. Boccaccini was not part of the research. The coauthor relationship of authors from different organizations was analyzed from Table 3.2. Table 3.2 enlists the organizations from where at least five articles were published. It is evident that the authors of the University of Tehran and the Tehran University of Medical Sciences collaborated. Similarly, the authors



CHAPTER 3 Biopolymer-based composites

Table 3.2 Coauthorship analysis based on the author’s organization. #





Total link strength

1 2

University of Tehran Tehran University of Medical Sciences University of the Basque Country Politehnica University of Bucharest Indian Institute of Technology University of Minho

Iran Iran

7 5

127 215

2 2

















3 4 5 6

from the University of the Basque Country and the Politehnica University of Bucharest collaborated. The number of articles from the University of Tehran and the Politehnica University of Bucharest was higher than the Tehran University of Medical Sciences and the University of the Basque Country, respectively. This is suggestive of the absence of collaboration among the authors of the paired organizations in some cases. Subsequently, the coauthorship analysis based on the country of affiliation of the authors was carried out. The summary of the analysis is tabulated in Table 3.3. During the analysis, countries with at least 15 articles were taken into consideration. It can be seen that the collaboration of the authors from the United States was the highest with the authors from other countries. A careful observation suggests that the number of articles from the authors of the United States was comparatively lower than or equal to those from the authors from countries like the Peoples’ Republic of China, India, and Iran, respectively. But the collaboration of the authors was in the order of Peoples’ Republic of China, Iran, and India, respectively. The software segregated the countries into two groups (Fig. 3.5). In the first group, there were five countries, namely the United States, Peoples’ Republic of China, Iran, India, and Malaysia. The other group consisted of the countries Spain and Brazil.

3.2.2 Cooccurrence analysis The analysis of the cooccurrence of the keywords was carried out. The software detected 1858 keywords from the downloaded bibliometric data. For the analysis, the keywords that appeared at least five times were selected. This narrowed down the number of keywords to 134. Henceforth, the names of the biopolymers were chosen manually. It was found that there were 11 biopolymers that have been explored for the development of composites for drug delivery applications. Postidentification of the keywords, the bibliometric data were used to obtain the

3.2 Scientometric analysis

Table 3.3 Coauthorship analysis based on the author’s country. #




Total link strength

1 2 3 4 5 6 7

United States Peoples’ Republic of China Iran India Malaysia Spain Brazil

37 52 37 48 15 22 18

1464 870 961 821 246 916 392

20 15 12 11 10 10 6

FIGURE 3.5 Coauthorship analysis based on the author’s country.

Density Visualization plot (Fig. 3.6). The visual analysis of the Density Visualization plot suggested that biopolymers like chitosan, alginate, cellulose, and hyaluronic acid are closely related to drug delivery applications. Accordingly, the properties of the above-mentioned polymers will be discussed briefly in this section. Chitosan Chitosan is synthesized by the partial deacetylation of chitin, the second-most abundant naturally occurring polymer. In other words, chitosan is not available in nature. It is derived by biological or chemical modification of chitin. Herein, it is noteworthy to mention that cellulose is the most abundant natural polymer. Chitin is extracted from various natural sources, including the cell walls of fungi, shrimps, crabs, and insects (Fig. 3.7; da Silva Alves, Healy, Pinto, Cadaval, & Breslin, 2021). However, the main sources of chitosan are shrimps and crabs. The chemical structures of chitin and chitosan have been provided in Fig. 3.8 (Younes & Rinaudo, 2015). Both chitin and chitosan are linear polysaccharides. Chitosan carries a positive charge when solubilized in acidic solutions (Matica, Aachmann,



CHAPTER 3 Biopolymer-based composites

FIGURE 3.6 Cooccurrence analysis of the keywords from the bibliometric data.

FIGURE 3.7 (A) Sources of chitin and (B) methods of preparation of chitosan. Reproduced from da Silva Alves, D. C., Healy, B., Pinto, L. A. D. A., Cadaval, T. R. S. A., & Breslin, C. B. (2021). Recent developments in chitosan-based adsorbents for the removal of pollutants from aqueous environments. Molecules (Basel, Switzerland), 26(3). under Creative Commons License.

3.2 Scientometric analysis

FIGURE 3.8 Chemical structure of chitin and chitosan. Reproduced from Younes, I., & Rinaudo, M. (2015). Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs, 13(3), 1133 1174. under Creative Commons License.

Tøndervik, Sletta, & Ostafe, 2019). Accordingly, chitosan is regarded as a polycationic polymer. Chemically, chitosan is composed of two subunits, namely, β-(1 4)-linked D-glucosamine and N-acetyl-D-glucosamine (Cheung, Ng, Wong, & Chan, 2015). These subunits are randomly arranged throughout the polymer skeleton. The polymer has found applications in various fields of study like food packaging, tissue engineering, drug delivery, wound healing, and bioremediation. The main advantage of chitosan is its biodegradability. It is also nonimmunogenic and hence does not elicit immunological reactions. The biodegradable products of chitosan do not trigger an inflammatory reaction (Zhao et al., 2018). The matrices of chitosan promote mammalian cell attachment, migration, and differentiation (Huang et al., 2015). This suggests that chitosan is not only a biocompatible polymer but also can promote cell proliferation. Further, due to the polycationic nature of chitosan, it has inherent antimicrobial properties. The antimicrobial properties of chitosan are against not only the Gram-positive and Gram-negative bacteria but also fungi (Kong, Chen, Xing, & Park, 2010). The mechanisms of antimicrobial activities of chitosan are summarized in Fig. 3.9 (Ke, Deng, Chuang, & Lin, 2021). Alginate Like chitosan, alginates are also linear polysaccharides. However, unlike polycationic chitosan, alginates are polyanionic. Alginates can be readily solubilized in water. Hence, the polymer matrices of alginates swell when immersed in aqueous mediums. It is mainly obtained from the cell walls of brown algae (Rabille´ et al., 2019). Extraction of alginates from some bacterial strains (e.g., Pseudomonas and Azotobacter) has also been reported. There are two subunits that are arranged in block-like patterns that are organized either homogeneously or heterogeneously. The subunits of the alginates include 1,4-linked β-D-mannuronic acid (M) and 1,4



CHAPTER 3 Biopolymer-based composites

FIGURE 3.9 Antimicrobial activity of chitosan: (A) Gram-positive bacteria, (B) Gram-negative bacteria, and (C) fungi. Reproduced from Ke, C.-L., Deng, F.-S., Chuang, C.-Y., & Lin, C.-H. (2021). Antimicrobial actions and applications of chitosan. Polymers, 13(6). under Creative Commons License.

α-L-guluronic acid (G) monomers (Fig. 3.10). If the M and G subunits are present heterogeneously, then the blocks are regarded as MG blocks. On the contrary, poly-M and poly-G blocks are formed in the alginates if the subunits are homogenously present (Barbu et al., 2021). These subunits may be arranged in three possible ways, viz., consecutive G subunits, consecutive M subunits, and alternating MG or GM subunits (Sun & Tan, 2013). Since the polymer is polyanionic, it can be ionically crosslinked with positive ions like calcium ions and different metal ions (e.g., cobalt ions, manganese ions, etc.) (Fig. 3.10; Dodero et al., 2019). The polymer is inherently biocompatible and biodegradable. Some of the properties of alginates are summarized in Fig. 3.11 (Gheorghita Puscaselu, Lobiuc, Dimian, & Covasa (2020). The properties of the alginate matrices can be enhanced multifolds through physical and chemical methods. The affinity of the mammalian

3.2 Scientometric analysis

FIGURE 3.10 (A) Chemical structure of alginates and (B) mechanism of ionic crosslinking using calcium ions (herein, M stands for calcium ions). Reproduced from Dodero, A., Pianella, L., Vicini, S., Alloisio, M., Ottonelli, M., & Castellano, M. (2019). Alginate-based hydrogels prepared via ionic gelation: An experimental design approach to predict the crosslinking degree. European Polymer Journal, 118, 586 594 under Creative Commons License.

FIGURE 3.11 Properties of alginates. Reproduced from Gheorghita Puscaselu, R., Lobiuc, A., Dimian, M., & Covasa, M. (2020). Alginate: From food industry to biomedical applications and management of metabolic disorders. Polymers, 12(10). https:// under Creative Commons License.



CHAPTER 3 Biopolymer-based composites

cells toward the alginate matrices can be tuned by affixing various ligands like peptide and sugar molecules as pendant groups. Functionalization of the alginates can also allow us to tune the biological properties of the alginate matrices (Dalheim et al., 2016). One such method of functionalization of alginates is shown in Fig. 3.12 (Huamani-Palomino, Co´rdova, Pichilingue L, Venaˆncio, & Valderrama, 2021). Further, the blending of alginates with other polymers has been proposed to improve the properties of the alginate matrices. Cellulose Cellulose is the most abundant polysaccharide in nature and is an organic compound. It is inherently biocompatible and biodegradable. The polysaccharide can be easily found in the cell walls of green plants (Mihranyan, 2011). It can also be extracted from various algae. Chemically, it is a polymer of D-glucose molecules. The process of polymerization is attained by the formation of β(1-4)-glycosidic bonds among the glucose molecules (Fig. 3.13; Tayeb, Amini, Ghasemi, & Tajvidi, 2018). The polymerization of the glucose molecules occurs during the replication of the plant cells. The formed polysaccharide, that is, cellulose, is a linear-chain polysaccharide like chitosan and alginates. In nature, cellulose exists

FIGURE 3.12 Functionalization of sodium alginate by oxidation and reductive amination. Reproduced from Huamani-Palomino, R. G., Co´rdova, B. M., Pichilingue L, E. R., Venaˆncio, T., & Valderrama, A. C. (2021). Functionalization of an alginate-based material by oxidation and reductive amination. Polymers, 13(2). under Creative Commons License.

3.2 Scientometric analysis

FIGURE 3.13 Chemical structure of cellulose. Reproduced from Tayeb, A. H., Amini, E., Ghasemi, S., & Tajvidi, M. (2018). Cellulose nanomaterials— Binding properties and applications: A review. Molecules (Basel, Switzerland), 23(10). 10.3390/molecules23102684. under Creative Commons License.

FIGURE 3.14 Mechanism of interconversion of the cellulose polymorphs. Reproduced from Naomi, R., Bt Hj Idrus, R., & Fauzi, M. B. (2020). Plant- vs. bacterial-derived cellulose for wound healing: A review. International Journal of Environmental Research and Public Health, 17(18). https:// under Creative Commons License.

only as type-I and type-II polymorphs, even though other polymorphs have also been proposed (Bian, Yang, & Tu, 2021). Fig. 3.14 depicts the mechanism of interconversion of the polymorphs. In recent years, cellulose of bacterial origin has also been reported by many researchers. In recent years, cellulose of bacterial origin has also been reported by many researchers. Bacterial cellulose is synthesized by the bacteria of the genera Gluconacetobacter, Agrobacterium, and Sarcina (Naomi, Bt Hj Idrus, & Fauzi, 2020). The mechanism of cellulose synthesis by the bacteria is summarized in Fig. 3.15. Since cellulose has many hydrophilic hydroxyl groups, cellulose is innately hydrophilic. A single unit of the glucose molecule in the cellulose backbone consists of three hydroxyl groups. However, as cellulose is a macromolecule, it is not soluble in water or aqueous solutions. Additionally, it is also not soluble in many organic solvents. It is easy to chemically modify the cellulose structure that has been associated with the chemistry of cellulose. The modification can be achieved without compromising the natural property of the polymer. The cellulose chain length, which plays a significant role in governing the cellulose matrices’ physical properties, depends on the cellulose source (Costa et al., 2019). The cellulose that is obtained from wood



CHAPTER 3 Biopolymer-based composites

FIGURE 3.15 Mechanism of synthesis of bacterial cellulose. Reproduced from Naomi, R., Bt Hj Idrus, R., & Fauzi, M. B. (2020). Plant- vs. bacterial-derived cellulose for wound healing: A review. International Journal of Environmental Research and Public Health, 17(18). https:// under Creative Commons License.

pulp may contain 300 1700 units of glucose molecules. On the other hand, bacterial cellulose may consist of 800 10,000 units of glucose molecules. Hyaluronic acid Hyaluronic acid is a glycosaminoglycans-based natural polymer. It is an unbranched heteropolysaccharide. The presence of the polysaccharide can be widely found in nature. Hyaluronic acid is not only found in humans and animals but also in algae, yeasts, and bacteria (Fallacara, Baldini, Manfredini, & Vertuani, 2018). The polysaccharide is composed of two subunits, namely N-acetyl-D-glucosamine and D-glucuronic acid, which are connected through ß-1,3-glycosidic linkages. Fig. 3.16 depicts the chemical structure of hyaluronic acid. The figure also depicts the hydrophilic and hydrophobic groups that are present in the hyaluronic acid chain. Additionally, the formation of hydrogen bonds by the polysaccharide in an aqueous solution has been shown. These hydrogen bonds stabilize the secondary structure of hyaluronic acid. A combination of the hydrophobic interactions and the hydrogen bonds causes the formation and stabilization of ßsheet tertiary structure in the aqueous solutions of hyaluronic acid. From the chemical structure of hyaluronic acid, it is clear that hyaluronic acid is a polyelectrolyte. Accordingly, the physical properties of the hyaluronic acid matrices are governed by the pH and ionic strength. The chemical modification of hyaluronic

3.2 Scientometric analysis

FIGURE 3.16 (A) Schematics of the chemical structure of hyaluronic acid, and (B) demarcation of hydrophilic groups, hydrophobic groups, and hydrogen bonds within the hyaluronic acid polymer chain. Reproduced from Fallacara, A., Baldini, E., Manfredini, S., & Vertuani, S. (2018). Hyaluronic acid in the third millennium. Polymers, 10(7). under Creative Commons License.

acid is straightforward and allows to tailor of the physical and chemical properties of the hyaluronic acid as per the requirement. Fig. 3.17 summarizes the possible ways for chemical modifications of hyaluronic acid and the polysaccharide types used in the pharmaceutical industries.

3.2.3 Analysis of the citations of the articles The articles were then analyzed for their citations. The articles that have received at least 100 citations were selected. The list of such articles is tabulated in Table 3.4. In the present section, the summary of the research works in the selected articles will be discussed.



CHAPTER 3 Biopolymer-based composites

FIGURE 3.17 (A) Schematic representation of the ways to modify the chemical structure of hyaluronic acid, and (B) types of hyaluronic acid used in pharmaceutical industries. Reproduced from Fallacara, A., Baldini, E., Manfredini, S., & Vertuani, S. (2018). Hyaluronic acid in the third millennium. Polymers, 10(7). under Creative Commons License.

Leach and Schmidt (2005) have reported the synthesis of photocrosslinkable hyaluronic acid polyethylene glycol hydrogel as tissue engineering scaffolds. The developed scaffolds were used as delivery matrices for the proteins. The hyaluronic acid biopolymer was chosen in the study because of its inherent biocompatibility and nonimmunogenicity. Chemically, the biopolymer is a glycosaminoglycan, which is a nonadhesive polysaccharide. Additionally, biopolymer also plays an important role in various biological processes. Some of the notable biological processes include its ability to modulate angiogenesis and the inflammatory response. The authors reported that they have previously developed a photocrosslinkable derivative of hyaluronic acid, namely photopolymerizable glycidyl methacrylatehyaluronic acid. The derivative was found to be biocompatible. The derivatization of the biopolymer allowed the authors to control the degradation rates of the polymeric constructs of the derivatized biopolymer. Most importantly, the properties of

3.2 Scientometric analysis

Table 3.4 List of documents that have received at least 100 citations. #





Characterization of protein release from photocrosslinkable hyaluronic acidpolyethylene glycol hydrogel tissue engineering scaffolds Intrathecal delivery of a polymeric nanocomposite hydrogel after spinal cord injury Biodegradable and thermo-sensitive chitosan-g-poly(N-vinylcaprolactam) nanoparticles as a 5-fluorouracil carrier Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications Pectin/carboxymethyl cellulose/ microfibrillated cellulose composite scaffolds for tissue engineering Electrophoretic deposition of gentamicinloaded bioactive glass/chitosan composite coatings for orthopedic implants Fabrication of bio-nanocomposite films based on fish gelatin reinforced with chitosan nanoparticles


Leach and Schmidt (2005)


Baumann, Kang, Tator, and Shoichet (2010)



Rejinold, Chennazhi, Nair, Tamura, and Jayakumar (2011) Coimbra et al. (2011)


Ninan et al. (2013)


Pishbin et al. (2014)


Hosseini, Rezaei, Zandi, and Farahmandghavi (2015)







the derivatized biopolymer could be tailored using peptide moieties. The authors reported that the hydrogels of pristine derivatized biopolymer and derivatized biopolymer/polyethylene glycol were suitable for the release of the protein molecules. In the study, bovine serum albumin was used as the model protein. The authors reported that the hydrogel developed with 1% of the derivatized biopolymer could quickly release the model protein. It was observed that .60% of the model protein was released within 6 h. Interestingly, an increase in the concentration of the derivatized biopolymer or polyethylene glycol delayed the release of the model protein. The analysis of the released protein molecules confirmed that the developed hydrogels did not alter the native monomeric form of the model protein. Lastly, the authors reported that the release of the model protein was prolonged to several weeks when the model protein was incorporated within the poly(lactic-co-glycolic acid) microspheres, which were incorporated afterward within the hydrogels. The authors concluded in their study that the novel photopolymerizable composites were suitable for the delivery of proteins in their active form and could be explored successfully for tissue engineering applications. Traumatic spinal cord injury (SCI) is a severe type of injury that primarily results in the life-long disability of the patients. A typical SCI may induce lasting



CHAPTER 3 Biopolymer-based composites

paralysis lower than the position of injury. To date, there is no cure for SCI. However, various researchers have reported that molecular medicine based therapy has shown great success in animal models. The primary aim of the treatment of SCI is to prevent degeneration of the functional tissue and rejuvenate the functionality of the already degenerated tissues to the extent possible. In this regard, Baumann et al. (2010) have reported the local delivery of the bioactive agents directly to the site of action. The use of such a delivery system allows circumventing the blood spinal cord barrier. This delivery system is essential because most bioactive agents cannot permeate through the blood spinal cord barrier and cannot elicit therapeutic activity. The matrix of the delivery system was developed using a biopolymeric combination of hyaluronan and methylcellulose. The developed matrix was an injectable hydrogel that could be quickly injected within the intrathecal space. The incorporation of the poly(lactic-co-glycolic acid) nanoparticles altered the rheological properties of the biopolymeric hydrogels. This observation was explained by the increased hydrophobic interactions between the methyl groups of methylcellulose and the nanoparticles. Such an increase in the rheological properties was related to the better stability of the composite hydrogels. Further, it was found that the developed hydrogels were well tolerated within the intrathecal space of the spinal cord of rats. The microglial activation was also quite limited in the presence of the hydrogels. The incorporation of the composite hydrogel within the healthy rats did not affect their locomotor function. The use of thermo-sensitive nanoparticles has been used for cancer treatment. The anticancer drugs are encapsulated within the thermo-sensitive nanoparticles, which are then directly injected into the bloodstream. Such drug delivery systems are expected to deliver the drugs directly to the tumor site. Rejinold et al. (2011) have reported the synthesis of nanoparticles for the treatment of cancer. Composite nanoparticles were synthesized using chitosan-g-poly(N-vinylcaprolactam). The synthesis of the nanocomposite was achieved by the ionic crosslinking method. 5-Fluorouracil, a well-established anticancer drug, was incorporated within the nanocarrier. The drug molecules formed intermolecular hydrogen bonding with thermo-responsive chitosan-g-poly(N-vinyl caprolactam). The in vitro drug release studies divulged an increased amount of drug release above 38 C, which was the lower critical solution temperature for the nanocomposite matrix. It was observed that the cancer cells were capable of uptaking the 5fluorouracil containing nanoparticles, thereby eliciting anticancer activity. The authors concluded that the developed composites were suitable for exploring as a delivery vehicle in cancer treatment. Composite scaffolds have found applications in tissue engineering. Highly porous scaffolds are used considerably in tissue engineering applications. The scaffolds help in the three-dimensional regeneration of the cells to develop targeted tissues and are capable of delivering bioactive agents like growth factors and drugs. One such application is bone tissue engineering. Coimbra et al. (2011) have developed a polyelectrolyte complex scaffold using the polysaccharides

3.2 Scientometric analysis

pectin and chitosan for possible bone tissue engineering. The polyelectrolyte complex is formed when polycationic and polyanionic polymers are mixed. Such polyelectrolyte complex systems have also been explored as drug delivery systems. Herein, pectin is a polyanionic polymer, and chitosan is a polycationic polymer. In the study, pectin derived from citrus fruits was used, while the chitosan used in the study was derived from crab shells. The aqueous solutions of the polysaccharides were used to develop the polyelectrolyte complex. During the preparation of the polyelectrolyte complex, the pH was maintained at 4.5. Thereafter the hydrogels were converted to scaffolds by the freeze-drying method. The elemental analysis of the scaffolds suggested the presence of both chitosan and pectin. This suggests that both the polymers retained their chemical identity in the scaffolds. The scaffolds were highly porous, but the pores were irregular in shape and size. Further, it was observed that the mass of the developed scaffolds was reduced to half when immersed in phosphate buffer solution (pH 7.4) for 1 month. Human osteoblast cells were able to adhere and proliferate over the scaffolds suggesting the biocompatibility and noncytotoxic nature of the developed scaffolds. The authors concluded that the scaffolds could be incorporated with bioactive inorganic materials (e.g., hydroxyapatite) to develop composite scaffolds. Ninan et al. (2013) have reported the synthesis of composite scaffolds of pectin, carboxymethyl cellulose, and microfibrillated cellulose for tissue engineering applications. The scaffolds were also prepared by the freeze-drying (lyophilization) method. The crosslinking of the composite matrix was carried out by the ionic crosslinking method. For crosslinking, a calcium chloride solution was used. It was observed that the porosity of the scaffolds was dependent on the composition of the scaffold matrix. The pore sizes of the developed scaffolds were in the range of 10 250 μm. Such pore sizes have been reported to be sufficient enough for tissue engineering applications. The crystalline lattice planes of microfibrillated cellulose could be deciphered within the composite scaffolds by the X-ray diffraction (XRD) study. The incorporation of microfibrillated cellulose within the scaffolds improved the thermal stability and reduced the degradation rate of the composite scaffolds. The composite scaffolds were also found to improve the cell viability of the NIH3T3 cells, which are acquired from mouse embryonic fibroblasts. The metallic implants are nonbioactive. Such implants may be made bioactive by coating the implants with bioactive materials. These implants have been proposed as a long-term solution for treating bone defects that are larger than a critical size. In this regard, Pishbin et al. (2014) has proposed electrophoretic deposition of glass and chitosan-based multifunctional composite coatings. The coatings were also loaded with gentamicin, an antibiotic drug. The electrophoretic deposition technique allowed the researchers to deposit glass, chitosan, and gentamicin onto a stainless steel substrate in a single step. This helped the researchers to reduce the complexity of the coating process. The coating allowed the deposition of hydroxyapatite over the coated stainless steel substrate, indicating its bioactivity. Also, the proliferation of the MG-63 cells, osteoblast-like cells, was promoted. Gentamicin was released in a sustained manner from the coatings. In



CHAPTER 3 Biopolymer-based composites

the first 5 days, 40% of the gentamicin was released from the coated substrate. The gentamicin-loaded coatings showed antimicrobial activity against Staphylococcus aureus. The authors concluded that the developed method would help the implant manufacturers to coat the metallic implants efficiently. Hosseini et al. (2015) have reported the synthesis of chitosan nanoparticles by the ionotropic gelation method. Sodium tripolyphosphate was used as the ionic crosslinker for the chitosan molecules. The prepared nanoparticles were spherical and had sizes in the range of 40 80 nm. The zeta potential of the synthesized nanoparticles was 110 mV. Subsequently, the nanoparticles were used to develop nanocomposites wherein fish gelatin was used as the polymer matrices. The distribution of the nanoparticles within the gelatin matrix was homogenous when the filler content was low. However, at higher filler content, agglomeration of the filler nanoparticles was observed. Infrared spectroscopy suggested that hydrogen bonding was prevalent among the chitosan nanoparticles and the gelatin matrices. The inclusion of the nanofillers improved the mechanical stability of the films but consequently reduced the elongation at break. The composite films could significantly improve the ultraviolet barrier and the water vapor permeability properties. The results suggested that the nanocomposite films were superior to the pristine gelatin film.

3.3 Conclusion There has been continuous development in designing composite biopolymers over the last two decades, specifically in the field of drug delivery. Nontoxic, biodegradable, and biocompatible nature of natural polymers are a few attributes that support their constant development. The natural abundance of biopolymers allows its maximum utilization for controlled delivery systems. The biocomposite polymeric system can prevent drugs/bioactive agents from the harsh microenvironment until it reaches the targeted site. The current chapter made an effort to perform a qualitative and quantitative evolution of biocomposites for drug delivery applications over time through scientometric study. Analysis of the data was carried out using freeware, VoSviewer software. The data obtained from the density visualization plot of the cooccurrence keywords suggested chitosan, alginate, cellulose, and hyaluronic acid are closely associated composite-based drug delivery systems. The chapter covered a brief description of these biopolymers and the possibility of their structural modification to serve efficiently as drug delivery systems. In the final section of the chapter, we listed a few of the most-cited literature in the field and tried to summarize those research works.

References Balart, R., Garcia-Garcia, D., Fombuena, V., Quiles-Carrillo, L., & Arrieta, M. P. (2021). Biopolymers from natural resources. Polymers, 13(15). Available from 10.3390/polym13152532.


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CHAPTER 3 Biopolymer-based composites

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Characteristics and characterization techniques of bacterial cellulose for biomedical applications—a short treatise


Kumar Anupam1,2, Richa Aggrawal3, Jitender Dhiman4, Priti Shivhare Lal5, Thallada Bhaskar6,7 and Dharm Dutt1 1

Department of Paper Technology, Indian Institute of Technology Roorkee, Saharanpur, Uttar Pradesh, India 2 Chemical Recovery and Biorefinery Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India 3 Department of Chemical Engineering, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, Haryana, India 4 Biotechnology Division, Central Pulp, and Paper Research Institute, Saharanpur, Uttar Pradesh, India 5 Physical Chemistry, Pulping and Bleaching Division, Central Pulp and Paper Research Institute, Saharanpur, Uttar Pradesh, India 6 Material Resource Efficiency Division, CSIR-Indian Institute of Petroleum, Dehradun, Uttarakhand, India 7 Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, Uttar Pradesh, India

4.1 Introduction With the advancement in medical science there emerges a great demand of biomaterials. Biomaterials possess great importance in the field of surgery. About 2800 years ago, before the evolution of modern medical science, great Indian surgeon Sushruta innovated medical surgery and utilized sutures made up of cellulose extracted from hemp and cotton. With modern techniques cellulose was extracted in 1838 from the cell walls of plants (Bodin, Ba¨ckdahl, Petersen, & Gatenholm, 2011; Hestrin & Schramm, 1954). Cellulose is a polymer which is made up of β-(1, 4) glucose and responsible for structural integrity of various fungi, plants, and some algae. Cellulose obtained using bacteria is called as bacterial cellulose (BC) and is in its pure form; whereas cellulose obtained from plants

Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 4 Characteristics and characterization techniques

also accommodate both lignin and hemicellulose in it. Physical and chemical properties of BC and plant based cellulose are same as both own same chemical structure. Bacterial species belonging to the Acetobacter, Rhizobium, Agrobacterium, and Sarcina genus are known to produce BC (Abol-Fotouh et al., 2020; Slapˇsak, Cleenwerck, De Vos, & Trˇcek, 2013; Trˇcek & Barja, 2015; Vigentini et al., 2019). In a discovery by Brown in 1886, it was found that bacterium Acetobacter xylinum produce cellulose in the form of film. At that time these bacterium was called as “the vinegar plant” because these were the chief source of acetic acid. In the middle of 20th century, research community became curious about BC. Hestrin and Schramm (in 1954) produced microbial cellulose using A. xylinum bacterium in glucose-plentiful medium. Because of the purity and excellent physicochemical characteristics BC can be utilized in the food and medical field. BC exhibit properties which are congruent in combining surface and macro-molecular characteristics that are important in biomedical field. During the synthesis of BC large quantity of water was trapped in its molecular structure, leading to the emergence of hydrogel possessing about 90% of water. BC was utilized as a noble wound dressing material due to inestimable in vivo biocompatibility, flexibility, higher water-holding capacity, and gas exchange (Czaja, Krystynowicz, Bielecki, & Brown, 2006; Sokolnicki, Fisher, Harrah, & Kaplan, 2006). Besides this, BC maintains a physical barrier that inhibits bacteria inflow in wound and only allows drug transportation in wounded region. Water present in the BC structure assists in wound healing as it provides necessary and consistent moisture supply to dry wound and inhibit necrosis. Characterization of necrosis in wound is carried out by blackness in wound. Such property is due to the dehydration and presence of death cells. Generally, healing of wounds is promoted by removing these dead cells from it; however, in crucial time when surgery is not possible, hydrogel dressing reinforced to serve the purpose (Abdelrahman & Newton, 2011; Kavitha et al., 2014). From the above discussion it is clear that BC holds great importance in biomedical filed. Therefore this chapter aims to reiterate and consider the applications of BC in biomedical field with major emphasis on physical, chemical, and biological properties of BC along with instrumental characterization techniques in light of their different morphologies and varieties.

4.2 Biomedical applications of bacterial cellulose This section discusses different types of characteristics and characterization techniques of BC as applied in various biomedical applications depicted in Fig. 4.1. An attempt has also been made to collate these characteristics and characterization techniques according to different morphologies and varieties of BCs reported in literature and shown in Fig. 4.2. Different characteristics and characterization techniques generally used for BCs are listed in Table 4.1.

4.2 Biomedical applications of bacterial cellulose

FIGURE 4.1 Bacterial cellulose implemented in various biomedical applications.

FIGURE 4.2 Morphologies and varieties of bacterial celluloses reported in literature.



CHAPTER 4 Characteristics and characterization techniques

Table 4.1 Characteristics and characterization techniques of BCs. Physical properties

Biological properties

Instrumental techniques

Tensile strength Hydrophobicity Water contact angle Surface tension Pore size/microporosity Electrical conductivity Thermal stability Optical properties Surface area Roughness Morphology Crystallinity Formability

Antimicrobial activity Cell viability Biocompatibility Biodegradability Cytotoxicity Cytocompatibility


AFM, atomic force microscopy; BC, bacterial cellulose; BET, Brunauer Emmett Teller theory; CP/MAS NMR, cross-polarization magic angle spinning nuclear magnetic resonance; DSC, differential scanning calorimetry; FTIR, Fourier transform infra-red; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TGA, thermo gravimetric analysis; XPS, X-photoelectron spectroscopy; XRD, X-ray diffraction; XRF, X-ray fluorescence.

4.2.1 Wound healing applications Bacterial cellulose-montmorillonite (BC-MMT) composites were used by UlIslam, Khan, and Park (2012a) to study its applications in biomedical field. Morphological study was performed on the composites using FESEM analysis. It revealed a 3D arrangement of microfibrils randomly arranged in BC matrix. MMT particles successfully penetrated deep inside BC sheets through pores thus confirming effective agglomeration of MMT particles on BC. FTIR spectrum of BC-MMT composites comprised a combination of FTIR spectra of PBC (pure bacterial cellulose) and pure MMT. The details of peaks are shown in Table 4.2. XRD patterns of pure BC resulted in three peaks at 2θ values of 14.2, 16.6, and 22.4 degrees indicating crystallographic planes of 110 ; (110) and (200), respectively. The pure MMT exhibited XRD peaks at 2θ values of 8.5, 17.74, 26.52, and 45.52 degrees. The XRD peaks of PBC and pure MMT altogether make up the XRD peaks for BC-MMT composites. TGA analysis revealed that the composites had a 5% weight loss over the 80 C 120 C temperature range because of moisture loss and coordinated water molecules between the layers. BC-MMT prepared with 1%, 2%, and 4% MMT suspensions exhibited that incorporation of MMT into BC improved its mechanical properties such as tensile strength and Young’s Modulus while strain and the water-holding capacity decreased. The variations in the values of these properties of pure BC with incorporation of MMT are shown in

4.2 Biomedical applications of bacterial cellulose

Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Material

FTIR spectrum peaks



O-H at 3444/cm, C-H at 2896/cm, and C-O-C at 1000/cm C-H at 1424/cm O-H at 3612/cm and Si-O at 1087/ cm H-O-H at 1600/cm and Si-O at 526/cm Hydrogen bonding at 3452/cm 3150/cm and 3280/cm for N-H; Peaks at 2924/cm for υas(CH2) and 2853/cm for υs(CH2) 1528/cm and 1697/cm υ (C 5 N), 3150/cm and 3280/cm N-H stretching vibrations. 2924/cm and 2853/cm υas(CH2) and υs (CH2), 3160, 3266, 2920, 2853, and 1618/cm for N-H, CH2 and C 5 N 3300/cm for OH, 2890/cm for CH and 1060/ cm for C-O-C, 1453/cm for CH and 1678/cm for H-O-H CH at 2895/cm, CH at 1428/cm and H-O-H at 1651 cm, 3342/cm for OH group CH at 2895/cm

Ul-Islam et al. (2012a)

3361/cm & 3235/cm for O-H, 2914/cm & 2850/cm for C-H, 1064/cm, 1116/cm, and 1236/cm for C-O-C and O-H 2897/cm for CH, 1032/cm and a carboxyl group band at 1595/cm 1646/cm for glucose carbonyl of cellulose, 1900 1500/cm for carbonyl and carboxyl groups, carboxyl group of BCA sponges of 70%, 50%, and 30% alginate were shifted from 1591/cm to 1608, 1610, and 1640/cm, respectively. O-H at 3300/cm, C-H at 2820/cm and C-O-C at 1080/cm and hydrogen bonded carbonyl group at 1730/cm, CH2 at 1427/cm and O-H at 684/cm

Tsai, Yang, Ho, Tsai, and Mi (2018)

Pure MMT


Pure BC


NR/BCW nanocomposite films SMN-Zein/ BC nanocomposite films Alginate/ BC hydrogel beads BCA sponge


Kukharenko et al. (2014)

Khalid et al. (2017)

Wahid et al. (2019)

Yin et al. (2018)

Kim et al. (2017) Chiaoprakobkij, Sanchavanakit, Subbalekha, Pavasant, and Phisalaphong (2011)

Zhijiang, Chengwei, and Guang (2012)




CHAPTER 4 Characteristics and characterization techniques

Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Continued Material

FTIR spectrum peaks

Pure P(3HB-co4HB)

C-O at 1283/cm, C-H at 2741/cm, 2889/cm & 2973/cm and C 5 O at 1726/cm, hydroxyl groups at 3433/cm PBC

Oliveira Barud et al. (2015)

Pure SF


3348/cm for O-H, 2940/cm & 2893/ cm for C-H and 1040/cm for C-O-C 1613/cm, 1550/cm and 1377/cm for amide I, II, and III groups Peaks of C-O at 1163/cm and C-O (asymmetric bridge) at 1061/cm, 1061/cm for C-O asymmetric bridge stretching & C-O-C for pyranose ring skeletal vibrations, intramolecular hydrogen bonding at 3200 3500/cm Bands of S 5 O (asymmetrical) at 1233/cm, phenyl connected to NH2 group at 1552/cm and NH2 at 3344/cm & 3391/cm both 1669/cm for C 5 O (amide I), 1634/ cm for C 5 O (A-ring), 1581/cm for C 5 O (C-ring), 1535/cm for NH2 deformation (amide II) and 1456/cm for C 5 C (aromatic ring) 2888/cm for C-H and 1159/cm for C-O (asymmetric bridge) Peaks at 3345/cm for hydroxyl groups and 1055/cm for C-O-C (pyranose skeletal ring vibration)

BC-Chi Pure BC

Pure AgSD




1040/cm for C-O (symmetric), 1168/cm for C-O-C (asymmetric), 2900/cm for C-H and 3500/cm for O-H, 400 700/cm for O-H, 1400/cm for CH2 and 1650/cm for H-O-H, 1340/cm for C-H deformation 1500 1600/cm for amide I and 1600 1700/cm for amide II, 1500 1600/cm and 1600 1700/cm for amide I and amide II, 1610 1630/cm for amide I and 1510 1520/ cm for amide II, 1640 1660/ cm and 1535 1542/cm represented silk I. Lin, Lien, Yeh, Yu, and Hsu (2013)

Shao, Liu, Wu, et al. (2016)

Shao, Liu, Wang, et al. (2016)

Zhang et al. (2020)


4.2 Biomedical applications of bacterial cellulose

Table 4.2 Functional groups present in different types of bacterial cellulose as evidenced from FTIR spectra. Continued Material

FTIR spectrum peaks

Pure TA

1710/cm for C 5 O and 1612/cm for C 5 C (aromatic), 1025/cm for benzene ring vibrations O-H at 3445/cm, C-H at 2920/cm & 1317/cm and C-O-C at 1157/cm, β-1,4 glycosidic bond at 1052/cm amide I at 1656/cm, amide II at 1540/cm, and amide III at 1245/cm amide I at 1653/cm and amide II at 1563/cm


Pure SS Pure HA


Wang, Tang, Huang, and Hui (2020)

BCW, bacterial cellulose whiskers; HA, hyaluronic acid; MMT, montmorillonite; NR, natural rubber; PHMG, polyhexamethylene guanidine hydrochloride; SMN, silymarin; SS, silk sericin; TA, tannic acid; TCH, tetracycline hydrochloride.

FIGURE 4.3 Effect of MMT incorporation on pure bacterial cellulose.

Fig. 4.3. The final inference derived from various characterizations suggested that BC-MMT composites can serve as excellent wound healing materials when used like a moist paste on wounds or skin. Kukharenko et al. (2014) combined BC with polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl) to prepare a material that can be utilized in wound healing. The material was characterized using FTIR, AFM,



CHAPTER 4 Characteristics and characterization techniques

antimicrobial assay, etc. The peaks identified through FTIR spectroscopy are mentioned in Table 4.2. AFM images displayed an interconnected network of microfibrils twisted bundles. Estimated diameter of these bundles was found to be 90 200 nm. Antimicrobial activity of BC/PHMG-Cl films revealed that PHMGCl was capable of inhibiting growth of all tested microorganisms. Inhibition zone of 20 mm was observed for gram-negative bacteria (Pseudomonas syringae and Klebsiella pneumonia) as well as gram-positive bacteria (S. aureus). Overall results concluded that BC/PHMG-Cl films are excellent and safe choice for wound healing. Bacterial cellulose-zinc oxide (BC-ZnO) nanocomposite films could be utilized extensively for wound healing applications. Khalid, Khan, Ul-Islam, Khan, and Wahid (2017) studied its effectiveness by subjecting the nanocomposite films to various characterizations. Morphological analysis of the nanocomposite films was carried out using FESEM analysis. SEM images suggested that ZnO nanoparticles were strongly attached to the surface of BC. Nanoparticles were found to be homogenously distributed and deeply penetrated throughout the BC matrix. It suggested the presence of enhanced thermal, mechanical, and biological properties. XRD patterns were obtained to determine the structural characteristics of the films. It was observed that XRD patterns of nanocomposite films totally comprised of peaks obtained from XRD patterns of BC and ZnO separately. Relative crystallinity of 52.4% was observed for nanocomposite films. The details of FTIR spectra of BC are provided in Table 4.2. FTIR spectra of BC-ZnO nanocomposite films contained all the peaks of FTIR spectra of BC and additionally had more peaks at 642 and 480/cm. Nanocomposite films displayed antibacterial activity against gram-negative bacteria such as Escherichia coli (90%), Citrobacter freundii (90.9%), and Pseudomonas aeruginosa (87.4%) and Gram-positive bacteria such as Staphylococcus aureus (94.3%). This suggested that ZnO nanoparticles were successfully incorporated on the surface of BC matrix and induced antimicrobial properties into BC making it potential for application in wound healing. Wound healing activity of nanoparticles resulted that wound size tended to shrink with passage of time. It was found to be 289 6 0 mm2 (day 0) to 98.3 6 7.6 mm2 (day 15). Another observation was made that 7%, 33%, and 66% healing took place on day 5, 10, and 15, respectively. Thus nanocomposite films displayed excellent healing capacities. It was concluded that BC-ZnO nanocomposite films have an excellent potential of application in burn wound healing. Khan, Ul-Islam, Ikram, et al. (2018) discovered the effective application of 3dimensional microporous regenerated bacterial cellulose/gelatin (3DMP rBC/G) scaffolds in wound healing and skin regeneration. Materials that are considered as ideal healing materials should be able to prevent deterioration of healing, prevent foreign body reactions, and be easily removed without damaging newly formed tissue. Hence different characterizations were applied to the scaffolds for analyzing their performance. Pores with high interconnectivity were found in the surface morphology of scaffolds. The cross-sectional morphology also displayed high content of gelatin entrapment in the scaffolds which led to enhanced cell

4.2 Biomedical applications of bacterial cellulose

interactions due to surface modification. Twenty four percent gelatin introduced into the scaffold was found to be used for strong hydrogen bonds between the OH and NH groups. The presence of gelatin improved the biocompatibility of 3-D scaffolds. Elemental analysis showed that pure BC was composed of C, H, and O, and gelatin was composed of C, H, O, and N. In NDBC samples, 51.31% oxygen, 42.51% carbon, 6.17% hydrogen, and 0% nitrogen were found. It was also suggested that introduction of gelatin into the scaffolds led to cellulose surface modification and enhanced biocompatibility. SEM images revealed good quality of cell adhesion and 3-D microporous structure which supported cell growth and proliferation because of availability of free space. High biocompatibility of 3-D scaffolds was supported because of the fact that cells penetrated deep inside the extracellular matrix and proliferated well with available free space. Gelatin was also found to be contributing to the biocompatibility of the scaffolds. Cell viability tests revealed that no difference was observed for the first 3 days while increased proliferation was observed after 5 7 days of incubation. Wound healing capabilities of 3-D scaffolds were found excellent after 2 weeks of application as the wound was almost completely closed. It was also observed that with time, new skin tissues grew in place of the scaffolds which led to successful skin regeneration. For 3DMP rBC/G scaffolds, wound healing rate was found to be 66.66 6 2.35% after 1 week which increased to 93.34 6 4.45% after 2 weeks. Histopathology analysis was performed to determine the healing progress and tissue regeneration phases. H&E-stained images resulted that successful reepithelialization takes place for 3-D scaffolds. H&E staining also proved increase in healing recovery. It was concluded that 3DMP rBC/G scaffolds have a great potential in wound healing applications. Performance of BC/ZnO nanocomposite films were well investigated by Wahid et al. (2019) for its applications in wound healing. FTIR, SEM, XRD, TGA were some of the characterizations applied to BC/ZnO-NP films. Characteristics of BC/ZnO-NP films were determined with the help of content and stability analysis, antibacterial activity, etc. XRD results provided with typical diffraction peaks at 14.5, 16.7, and 22.8 degrees attributed to (100), (010), and (110) crystallographic planes of cellulose. Moreover, peaks at 31.8, 34.4, 36.2, 48.5, 56.5, 63.2, and 68.4 degrees represented (100), (002), (101), (102), (110), (103), and (112) crystal planes of ZnO having hexagonal wurtzite structure. It was also observed that no peaks for impurities were found, therefore, indicating formation of BC/ZnO-NP films with no impurities. FESEM analysis suggested uniform distribution of ZnO-NPs on the surface of cellulose. FTIR results were found in correspondence with the results obtained from XRD and FESEM analysis. FTIR spectra gave various characteristic peaks as shown in Table 4.2. The peak at 438/cm was attributed to ZnO-NPs. The TGA results show that the nanocomposite film showed significant weight loss between 250 C and 330 C because of dehydration of absorbed water and thermal decomposition of BC. It was also suggested that BC/ZnO nanocomposite films possessed better thermal stability at elevated temperatures. The UV-visible spectra displayed a peak at 362 nm,



CHAPTER 4 Characteristics and characterization techniques

confirming existence of ZnO-NPs in the nanocomposite films. Photocatalytic activity revealed that the intensity of the peak at 464 nm reduced on enhancing the UV exposure time. It was estimated that almost 91% of MO was degraded by nanocomposite films. The antibacterial activity of nanocomposites was tested against Gram-positive as well as Gram-negative bacteria. The results discovered a sturdier consequence on Gram-positive bacteria (Bacillus subtilis) than Gramnegative bacteria (P. aeruginosa). It was concluded that introduction of ZnO-NPs onto BC surface successfully induced antibacterial properties into BC films. Bacterial cellulose/Bacillus subtilis (BC/BS) biocomposite was utilized by Savitskaya, Shokatayeva, Kistaubayeva, Ignatova, and Digel (2019) for its potential applications in wound healing. There are three major steps involved in destruction of bacteria through development of pores inside the BM (bacterial membrane). These are (1) binding to BM, (2) accumulation within BM, and (3) creation of channels. Various characterizations were applied to the biocomposite to determine its wound healing capability. SEM images revealed porous and microfibrillar structure of BC films which was highly suitable for impregnation of BS cells. Hence, SEM micrographs established effective agglomeration of BS P-2 cells to the surface of BC films. Antagonistic activity was first determined using agar diffusion test. The test confirmed antibacterial efficacy of this biocomposite as the inhibition zone was found to be in the range of 13 22 mm around fiber mats. Time kill test was also used to determine the antagonistic activity of biocomposite by revealing its bactericidal effect. Time kill test resulted in a time-dependent and concentration-dependent antimicrobial effect. It was observed that there was 100% reduction in bacterial growth in case of gram-negative bacteria in 24 h, while the same reduction was observed in just 10 h for gram-positive bacteria. Killing pattern of Staphylococcus epidermidis and S. aureus were found to be similar while good bactericidal effect was observed against E. coli and P. aeruginosa. The biocomposite was also studied for its proteolytic activity. It was suggested that BS agglomerated on BC matrix successfully produced proteases which were capable of digesting milk-casein. Average diameter of the zones was found to be 16 mm. It was suggested that the proteolytic enzymes produced by BS cells led to faster wound healing. Effective wound dressing is one that promotes epithelium regeneration. Dead tissues in contact of wound also promoted bacterial growth. Hence, it becomes mandatory for the wound healing material to be capable not only of inhibiting bacterial growth but also killing any further microbial growth. It was suggested that proteases produced with the help of BC/BS biocomposite supported healing of burned skin wounds, pressure sores, and leg ulcers. Study of epithelialization time and wound contraction were conducted on the biocomposite films. A very high wound healing activity was observed through BC/BS biocomposite films. Tests conducted on a group of rats displayed complete healing within a period of 7 days. It was found that epithelium effectively covered the wounds in moist environment. Hundred percentage of wound closure was achieved for animals treated with BC/BS biocomposite on seventh day. In conclusion, it was suggested that BS P-2 has advantageous and positive effect on wound healing.

4.2 Biomedical applications of bacterial cellulose

4.2.2 Diagnosis of ovarian cancer BC possesses extraordinary physicochemical as well as mechanical properties, however, it lacks anticancer activity. On the other hand, chitosan displays unique anticancer activity but has weak mechanical properties. Hence, Ul-Islam et al. (2019) employed 3-D scaffolds of bacterial cellulose and chitosan (BC-Chi) for its effective application in diagnosis of ovarian cancer. Usage of BC-Chi scaffolds led to improved mechanical and biological properties as well as utilization of anticancer activity. The scaffolds were characterized using a number of techniques including FTIR and FESEM analysis. The FTIR spectroscopy analysis confirmed the impregnation of chitosan onto the BC matrix which improved its mechanical properties and water-holding capacity along with delaying the water release rate from the matrix. These properties are desirable in an ideal scaffold to be effective in biomedical applications. SEM analysis revealed that BC matrix possessed a 3-D structure. Surface phase-contrast microscopic analysis was used in morphological studies which disclosed that A2780 cell lines had solid adhesive power towards cancer cells. Thus, strong adherence of cells on BC-Chi scaffolds suggested enhanced biocompatibility of scaffolds. An improvement in the adhesion and penetration of cell lines onto the scaffolds was observed through cross-sectional staining. WST-1 assay suggested high viability and proliferation. The H&E actin staining resulted in higher level of cell proliferation using BC-Chi scaffolds. This led to lower amount of cell aggregate formation indicating strong cell-scaffold interaction. Hence a comparatively high level of cell lines infiltration was observed. The results suggested BC-Chi scaffolds having great potential for in vitro culturing of cancer cell lines.

4.2.3 Shape memory material Adaptive medical implants, self-tightening sutures, self-retractable and removable stents, etc. are some of the applications of shape memory material in biomedical field. Utilization of natural rubber (NR) with bacterial cellulose whiskers (BCW) was explored by Yin et al. (2018) for its application in production of shape memory materials. NR/BCWs nanocomposite films were fabricated for this purpose through acid hydrolysis. This was done to retain crystalline parts to form nanowhiskers and to remove amorphous regions of BC. TEM, SEM, XRD, FTIR, etc. were some of the characterizations applied to nanocomposite films. The XRD patterns confirmed that the high crystallinity was due to the removal of amorphous regions of BC. The FTIR spectra peak has been shown in Table 4.2. This suggested the presence of intermolecular hydrogen bonds between cellulose chains. The morphology of BCWs suggested slender rod like shape of nanocomposite films. Mechanical properties indicated the development of a 3-D network of whiskers linked through hydrogen bonding and an abundance of immobilized rubber chains about the surfaces of filler. The water uptake capacity has a high diffusion coefficient of water because of the water diffusing along hydrophilic channels. This was due to high hydrophilicity of BCWs which led to an increase in hydrophilicity of composite films.



CHAPTER 4 Characteristics and characterization techniques

4.2.4 Preventing deterioration of salmon muscle and slowing down the lipid oxidation Tsai et al. (2018) explored the biomedical application using silymarin-zein (SMN-Zein) nanoparticles/BC nanocomposite films. The SMN-Zein nanoparticles were firmly attached to BC nanofibers. The FTIR spectrum of BC composite displayed characteristic bands shown in Table 4.2. The XRD diffraction patterns illustrated that crystallinity of BC fibers was not at all affected because of the interaction between SMN-Zein nanoparticles and BC fibers. Wetting properties of SMN-Zein/BC nanocomposite films suggested increase in the hydrophobicity of film surfaces. This causes packaging films to be moisture resistant which acts as an excellent obstruction against water penetration causing improved shelf life. The nanocomposite films demonstrated strong antibacterial activity against Grampositive bacteria such as S. aureus and was less effective against Gram-negative bacteria such as E. coli and P. aeruginosa.

4.2.5 Lipase immobilization Kim et al. (2017) investigated the potential of alginate/BC nanocomposite beads for lipase immobilization. The average diameter of alginate/BC hydrogel beads was found to be in the range of 2.5 3.6 mm. The average diameter and volume of alginate/BC hydrogel beads increased by 144% and 300%, respectively. This was attributed to swelling and high water-holding capacity of alginate/BC beads. SEM results revealed that BC was present inside the alginate beads until 24 h. After that, cellulose fibers were released to the bead surface. After cultivation time of 72 h, number of cellulose fibers and mats layered on the bead surface was found to be increased. The characteristic FTIR peaks are mentioned in Table 4.2. The results suggested that specific interactions take place between hydroxyl groups and carboxyl groups. The XRD patterns represented four peaks at 2θ 5 14.3 , 16.7 , 22.6 , and 34.5 , which corresponded to crystalline planes of cellulose. Alginate/BC nanocomposite beads also presented with an unreported peak at 2θ 5 24.0 which may represent formation of a new crystalline structure. The water vapor sorption capacity is responsible for high water-holding ability of alginate/BC nanocomposite beads. The water vapor sorption capacity was found to be 38.88 (g/g dry bead). Biodegradability of beads was supported with the fact that cellulose present inside the beads could be degraded by cellulase producing microorganisms. Thus, alginate/BC nanocomposite beads have a high potential in biomedical applications.

4.2.6 Tissue engineering Chiaoprakobkij et al. (2011) fabricated bacterial cellulose/alginate (BCA) composite sponges for effective utilization in biomedical field. The fabricated sponges were subjected to elemental analysis using X-ray fluorescence (XRF)

4.2 Biomedical applications of bacterial cellulose

spectroscopy. It resulted in absence of sodium (Na) peak suggesting complete removal of NaOH from BCA sponge. Also, calcium content of 0.88 2.96 weight % in the BCA sponges was observed due to alginate content. The peaks identified through FTIR spectra study are presented in Table 4.2. SEM micrographs revealed porous structure with 3-D interconnection throughout the sponges. It also told that asymmetric structure consisted of a top skin layer and sponge-like porous layer. The dense outer layer helps to prevent bacterial invasion and to avoid wound dehydration, whereas the porous layer provides drainage of wound and mechanical strength. Tensile strength and elongation at break were found to be decreasing with increasing the alginate content. Hence, it was inferred that intermolecular interactions might reduce the crystallinity and mechanical strength of the composite material. For water uptake ability, it was observed that BCA sponges swelled rapidly after immersion in water and were stable in distilled water. It was concluded that BCA sponge possessed exceptional characteristic properties for utilization in oral tissue regeneration. Poly(3-hydroxubutyrate-co-4-hydroxubutyrate) and bacterial cellulose biocomposite scaffolds [P(3HB-co-4HB)/BC] were engineered by Zhijiang et al. (2012) for tissue regeneration purposes. FESEM analysis revealed that scaffolds had multipore size distribution. Medium pores with diameter of 20 μm were uniformly dispersed on the surface. Micro pores with diameter 500 nm were found inside the wall. A 3-D network structure was obtained. Porosity of the scaffolds was found to be 91%. The findings of FTIR spectroscopy are shown in Table 4.2. All these characteristics bands and peaks of pure BC and pure P(3HB-co-4HB) together combined forms the FTIR spectra for P(3HB-co-4HB)/BC composite scaffolds. XRD analysis for pure P(3HB-co-4HB) resulted in two strong peaks at 2θ value of 13 and 17 degrees assigned to (020) and (110) orthorhombic unit cell. On the other hand, XRD pattern for P(3HB-co-4HB)/BC composite scaffolds is similar to XRD patterns of pure P(3HB-co-4HB) and cellulose II but with decreased intensity. Mechanical properties of P(3HB-co-4HB)/BC composite scaffolds suggested a visible yielding point but very close to breaking point. Values for tensile strength, elongation at break and Young’s modulus were found to be 46 MPa, 13.5% and 0.88 GPa respectively. The water contact angle for scaffolds was found to be reduced to 33.6 degrees and surface tension value was 59.2 mN/ m2. These results indicated high hydrophilicity of scaffolds. Scaffolds represented better biodegradability. After degradation for 30 days, weight loss ratio was 12% of original weight. It was concluded that the fabricated scaffolds were found suitable for tissue engineering uses. Favi et al. (2013) explored the potential of EqMSC (equine derived bone marrow mesenchymal stem cells)-BC scaffolds for applications in tissue regeneration. Scaffolds were subjected to MTS assay analysis to determine viability of EqMSCs on BC. It displayed extraordinary metabolic rates as a function time and denote a high viability and proliferation configuration. The linear regressions of the 1.0 3 104, 2.0 3 104, 3.0 3 104, and 4.0 3 104 cells seeded on BC were 0.998, 0.991, 0.966, and 0.950, respectively, indicating a linear response between cell



CHAPTER 4 Characteristics and characterization techniques

number and absorbance at 490 nm, further confirming the proliferation of EqMSCs under these conditions. Alkaline phosphatase staining of cell patterns confirmed that EqMSC-BC retained their structure and stem cell-like properties. SEM images revealed that EqMSC cells adhered to nanofibrous BC matrix and maintained a fibroblast membrane morphology. The study concluded that EqMSCs can adhere to, are metabolically active, viable, and retain the potential to differentiate into osteocytes and chondrocytes on BC matrix. Hyaluronic acid on bacterial cellulose (BC/HA) hybrid membranes were employed by Lopes, Riegel-Vidotti, Grein, Tischer, and Faria-Tischer (2014) for applications in tissue engineering. Different characterization techniques such as FTIR, XRD, NMR, AFM, etc. were applied to these membranes. XRD patterns of BC/ HA membranes displayed peaks at 14.6, 16.9, and 22.8 degrees referred to (100), (010), and (110) planes of cellulose I attributed to triclinic unit cell of allomorph Iα. It was suggested that membranes produced on third and sixth day after fermentation displayed highest crystallinity. Presence of HA was confirmed by 13C CP MAS NMR. The spectrum displayed both anomeric carbons of the glucuronic acid and the N-acetyl glucosamine at 101.6 ppm. Signals from HA were also observed through methyl group present at 23 ppm, N-acetyl present at 55 ppm, and carbonyl carbon present at 174 ppm. New signals observed at 30, 33, and 41 ppm were attributed to peptides/proteins. SEM micrographs exhibited a mesh like morphology of hybrid membranes. Surface morphology was also determined using AFM which revealed that fibrils were attached to each other in hybrid membranes. Wetting experiments revealed smoother and more hydrophilic surface. TGA analysis was carried out for a temperature range of 150 C 450 C over a period of 6 days. It was inferred that hybrid membranes were found to be more thermally stable. Yin, Stilwell, Santos, Wang, and Weibel (2015) explored the possibility of application of porous bacterial cellulose using agarose microparticles (pBC-M) in biomedical applications. Agarose can be termed as a polysaccharide which is extracted from red algae. Agarose has exceptional biocompatibility, physical and chemical stability and hydrophilicity. It can be used in biomolecule separation and microencapsulation. pBC-M characterized using SEM resulted in micrographs depicting a dense network of nanofibrous BC. For pBC-M micrographs, a uniform and homogenous distribution of pores with diameter 300 500 μm was observed. To analyze the feasibility of pBC-M in tissue engineering applications, surface of microparticle samples were cultivated using human P1 chondrocytes. Chondrocyte cells grew on the surface of BC and were not able to penetrate inside the polymer whereas these cells entered pBC-M scaffolds and dispersed throughout the polymer. This happened due to the smaller average pore size of the network as compared to cell dimensions. Chondrocytes cultivated on pBC-M scaffolds resulted in high cell viability of 85% 99% over a period of 14 days. Cell morphology of chondrocytes cultivated on the scaffolds was studied using confocal microscopy for 1, 7, and 14 days. After 1 day, cells formed cell body extensions while after 7 days, cells exhibited spindle shape indicating attachment

4.2 Biomedical applications of bacterial cellulose

to BC fibers. After 14 days, cells displayed a stretched morphology confirming even distribution throughout the porous scaffolds. Analysis of mechanical properties was performed for pure BC and pBC-M scaffolds. Values of Young’s Modulus and stress at break for pure BC were recorded to be 14.7 and 2.4 MPa and for pBC-M were recorded to be 5.4 and 0.52 MPa, respectively. It was suggested that values of mechanical properties of pBC-M were lying in the ideal range required for mechanical properties of materials used for tissue engineering. Thus, it was concluded that pBC-M can be a suitable material for cartilage repairing. Bacterial cellulose/silk fibroin (BC/SF) nanocomposites were developed by Oliveira Barud et al. (2015) for tissue engineering applications. The nanocomposites were subjected to various characterizations. The results of FTIR spectra study are shown in Table 4.2. FTIR spectra of BC/SF nanocomposites can simply be considered as summation of FTIR spectras of BC and SF separately. XRD peaks for pure BC were observed at 15 and 22.5 degrees assigned to native cellulose type 1. Freeze dried SF exhibited XRD peaks at 11.8, 19.8, and 22.6 degrees assigned to silk 1 crystalline plane. XRD patterns of BC/SF nanocomposites displayed no significant changes when compared to XRD patterns of BC and SF. BC, SF, BC/SF nanocomposites were all subjected to TGA analysis. Two mass losses were found in the TGA curve of BC. First mass loss was almost 4.6% which occurred at 200 C due to evaporation of surface water. Second mass loss was found to be 80% which occurred at 280 C due to decomposition and depolymerization of dehydrocellulose. Two mass losses were found in the TGA curve of SF. First mass loss was almost 7.3% which occurred at 120 C due to water loss. Second mass loss was found to be 52% which occurred at 180 C 500 C due to breakdown of amino acid residues and cleavage of peptide bonds. TGA curve of BC/SF nanocomposites represented three mass losses. First loss was almost 7% occurred at 200 C due to water losses. Other two occurred at 200 C 500 C. Images obtained from FESEM analysis displayed 3-D nano-fibril network of porous structure for BC matrix. SEM images for BC/SF: 25% and 50% nanocomposites revealed sponge-like structures whereas BC/SF: 75% nanocomposite exhibited less porous structure. Overall, a well interconnected porous structure of nano-filaments entangled with each other was observed as morphology for BC/SF nanocomposites. Pore size of the scaffolds were found to be 102 6 5.43 μm through porosity studies. Water solubility results obtained explained 0%, 3%, and 8.6% solubility of BC/SF: 25% nanocomposite, BC/SF: 50% nanocomposite and BC/SF: 75% nanocomposite in distilled water, respectively. Water uptake capacity is another important parameter for characterization. It discusses the property of a material to diffuse water which allows transport of nutrients and growth of new cells necessary for tissue regeneration. BC/SF: 50% nanocomposite scaffolds had a water uptake capacity of 216%. All scaffolds were able to absorb water within 1 min and get saturated within 1 h. Average cell viability for BC/SF: 50% nanocomposite was found to be 123.81%. No genotoxicity or cytotoxicity was found in the scaffolds which rendered it perfectly suitable for applications in tissue engineering.



CHAPTER 4 Characteristics and characterization techniques

Aki et al. (2020) employed 12 wt.% polyvinyl alcohol (PVA) /0.25 wt.% hexagonal boron nitride (hBN) /(0.1, 0.25, 0.5 wt.%) BC composite scaffolds for testing its applications in bone tissue engineering. The composite scaffolds were found suitable for biomedical applications after confirmation through various characterization techniques that were applied to the scaffolds. SEM micrographs revealed that 12 wt.% PVA composites exhibited a uniform and homogenous structure. Twelve weight percentage PVA/0.25 wt.% hBN composites were found to be having smooth surface. It was also observed that 12 wt.% PVA/0.25 wt.% hBN /0.5 wt.% BC composites had small pores of 265.68 6 15.39 μm suggesting successful vascularization and nutrient transport in tissue engineering applications. DSC thermographs suggested that 12 wt.% PVA composites were highly crystalline in nature because they had a melting point close to 230 C. It was also observed that addition of BC and hBN to 12 wt.% PVA composites caused a slight shift in the melting point of the composites. Physicochemical properties of various composites were also studied. Viscosity of PVA composites was found to be increasing with the addition of hBN and BC concentration. Twelve weight percentage PVA and 12 wt.% PVA/0.25 wt.% hBN composites possessed a 30% difference in viscosity values. No significant difference in the density values of the composites were found. It was found that an increment in BC concentration led to increase in the surface tension values. 0.127 6 0.05 MPa was observed as the highest value for tensile strength in case of 12 wt.% PVA/0.25 wt.% hBN/0.1 wt. % BC composite scaffolds. Values of elongation at break tended to increase with the introduction of additives to the various scaffolds. Another observation was made that BC and hBN caused increase in the ductile nature of the composites due to the hydrogen bonding. Maximum swelling degree was observed for 12 wt. % PVA composites. It was also suggested that highly rigid structure of BC caused increase in water absorption and permeation and limited the swelling behavior. Biocompatibility was verified using human osteoblast cells which resulted in increased proliferation and better extracellular matrix compatibility through 12 wt.% PVA/0.25 wt.% hBN/0.5 wt.% BC composite scaffolds. A reduction in the cell viability was observed on addition of BC concentration to PVA/hBN composites. It was concluded that the composite scaffolds have a great potential in bone tissue engineering applications.

4.2.7 Implantable devices in regenerative medicine Bacterial cellulose-gold nanoparticles (AuNPs)—poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) namely (BC-AuNPs-PEDOT:PSS) composites were successfully employed by Khan, Ul-Islam, Ullah, et al. (2018) for their potential application for implantable devices in regenerative medicine. FESEM, TEM, and AFM characterizations were applied for determination of size, structure, and arrangement of the composite. SEM images indicated a random arrangement of microfibrils which resulted in pore formation on the surface of BC matrix. It also displayed that PEDOT:PSS molecules completely filled up

4.2 Biomedical applications of bacterial cellulose

the empty spaces in BC-AuNPs nanocomposites. TEM images further confirmed the results obtained from SEM images while also discussing the fact that PEDOT: PSS acts as connecting sheets between the BC fibers. AFM analysis was used to investigate the topography of nanocomposites. A smooth topography is observed indicating completely filled up empty spaces between AuNPs and BC fibers by PEDOT:PSS. XRD technique is used to determine the crystalline features of nanocomposites. Lower crystallinity is observed in the composites due to the disturbances in the hydrogen bonding between BC chains. FTIR analysis is used to determine the functional groups and nature of bonds present in the composite. A broad OH peak was observed at 3627/cm referring to strong hydrogen bonding interactions between OH groups of BC and anions and cations of PEDOT:PSS. Water displacement was justified by lower intensity band of H-O-H observed at 1630 cm. FTIR spectra confirmed that the formation of composites was based on hydrogen bonding. High electrical conductivity of 16.65 6 1.27 S/cm was observed due to the penetration of PEDOT:PSS into the composites. Biocompatibility analysis suggested that successful adhesion of cells was observed after incubation. Even after 3 days, cells continued to grow and proliferate confirming filopodia formation and interconnection. Cytotoxicity analysis displayed no reduction in cell proliferation. This suggested that formation of composites does not cause any toxic effects, thus justifying the potential application of BC-AuNPs-PEDOT:PSS composites in implantable devices for biomedical applications.

4.2.8 Drug delivery Abeer, Amin, Lazim, Pandey, and Martin (2014) explored the potential of acrylated AbA-g-bacterial cellulose hydrogel (AcAA-g-BC) for its use in drug delivery applications. Numerous characteristic and characterization studies were carried out for the study. Solid state CP/MAS 13C NMR analysis was conducted on the hydrogel samples. Four hundred megahertz 13C NMR spectrum was observed for acrylated AbA-g-BC hydrogel. Peak at 62 ppm refers to C6 of anhydro-glucose unit of cellulose. It also represents substituted hydroxyl groups. Typical peaks at 72 and 74 ppm referred to C2, C3, and C5, whereas peaks at 82 and 89 ppm referred to C4 carbons. Peak at 107 ppm corresponded to C1 while peaks at 145, 124, 121, and 55 ppm corresponded to C2, C3, C4, and C5 carbons, respectively. The results obtained from NMR spectra confirmed the formation and successful grafting of hydrogel. SEM images concluded that at higher radiation doses, the pore network tends to decrease. This was attributed to the possibility of cross-linking at increased radiation doses and higher AA concentrations. A sponge-like morphology was observed for the hydrogels formed. It was also observed that the pore size obtained in this study was found similar to the pore size of hydrogels used traditionally in drug delivery. Gel fraction of hydrogel was found to be in the range of 73.0 6 1.0 to 83.0 6 1.9 and was attributed to the presence of AbA. Increasing radiation dose led to an increase in the gel fraction while it was not affected at all



CHAPTER 4 Characteristics and characterization techniques

on increasing concentration of AA. Swelling studies revealed that acrylated AbAg-BC hydrogel was pH responsive. Swelling ratio was found to be lowest at pH 2.0 and increased with pH. It also referred to potential application of hydrogels formed with controlled drug release at intestinal pH. It was observed from XRD analysis that peak at 2θ 5 16.5 degrees referred to crystallographic plane of BC while broad peak at 22.5 degrees referred to (002) plane. But in case of hydrogels, two different peaks at 2θ 5 10 and 16 degrees and a sharp peak at 22.5 degrees were observed. Characteristic peaks of BC were not available in the final hydrogel. Hence, it confirmed that AbA was effectively grafted onto hydrogel structure because of the decreased crystallinity. Differential scanning calorimetry analysis was done to find out the glass transition temperature (Tg). It was observed to be 80 C and was only available at endothermic peaks. The T value was higher because of the high molecular weight of hydrogels due to the presence of AbA. Also, another reason for high value of T was attributed to water resistance. Hence, it was suggested that AbA imparted water repellence to the hydrogel. Cell viability test revealed cell viability percentage to be 72% and it decreased with an increment in AA concentration. It was also suggested that AA concentration leads to acidic environment followed by cell destruction. On the concluding note, a great potential of the hydrogels formed was reported in drug delivery applications.

4.2.9 Bone healing Zimmermann, LeBlanc, Sheets, Fox, and Gatenholm (2011) examined the usage of mineralized BC for bone healing purposes. FESEM analysis revealed some interesting information regarding scaffolds. It was observed that tube out and pellicle BC scaffolds had many open surface morphologies which promoted crystal growth along BC fibrils. It was also observed that crystals were not able to completely cover up the fibrils. The tube in BC scaffold demonstrated a more extensive amount of crystallization on the surface. XRD pattern for pure BC had peaks at 2θ values of 14.5, 23.1, 29.4, 36.0, 39.4, 43.2, 47.5, 48.5, and 57.4 degrees, while XRD pattern for mineralized BC consisted of peaks at 14.4, 22.6, 27.4, 29.4, 31.8, 36.0, 39.4, 43.2, 45.5, 47.5, 48.5, and 56.5 degrees. Osteoprogenitor cell morphology illustrated that F-actin of cells formed a network over mineralized BC resulting in improvised adherence to the surface. This effect was not observed in pure BC. Various characterizations applied to mineralized BC supported its excellent usage in bone healing.

4.2.10 Wound dressing Single sugar α-linked glucuronic acid-based oligosaccharide combined with bacterial cellulose (SSGO/BC) composites were employed by Ul-Islam, Khan, and Park (2012b) for wound dressing applications. The composite was extensively characterized based on different concentrations of SSGO. FESEM analysis

4.2 Biomedical applications of bacterial cellulose

revealed the surface morphology of composite samples as reticulated fibril arrangement. The micrographs displayed that the fibrils were loosely arranged with larger pores of BC. It was also noted that thickness, density, and compactness increased with increment in SSGO concentration. BET analysis was performed to determine the pore size, pore volume and surface area of fabricated composites. The total surface area for BC0 was found to be 178 (m2/g) which decreased to 168 (m2/g) for BC1, 135 (m2/g) for BC2, and 104 (m2/g) for BC4. Similarly, total pore volume for BC0 was 0.505 cc/g which decreased to 0.144 (cc/g) for BC1, 0.124 (cc/g) for BC2, and 0.091 (cc/g) for BC4. Also, average ˚ for BC0 which decreased to 57.12 A ˚ for BC1, 58.06 A ˚ pore diameter was 309 A ˚ for BC2, and 49.48 A for BC4. Overall BET analysis suggested a decreasing trend in values with increasing SSGO content. Water-holding capacity was 106.43 times its dry weight for BC0 which decreased to 100.36, 91.84, and 85.31 times its dry weight for BC1, BC2, and BC4, respectively. Hence, water-holding capacity decreases with increasing SSGO content. The characteristics of SSGO/BC composites suggested its exceptional properties suitable for applications in wound dressing material. Lin et al. (2013) utilized BC combined with chitosan (BC-Ch) for wound dressing applications. Surface morphology of BC-Ch composites was assessed through SEM micrographs. It resulted in heavily compact membranes due to the presence of chitosan. It was suggested that chitosan may have penetrated into pores of BC forming a denser network and decreased pore size. The FTIR spectra identified various peaks as listed in Table 4.2. The FTIR spectra thus verified the presence of chitosan. Mechanical properties of BC-Ch composites were found to be 10 MPa, 29% and 132 MPa for tensile strength, elongation at break and Young’s modulus, respectively. This inferred adequate toughness of composite material for applications in wound dressing. Antibacterial activity was determined for the composites using gram-negative E. coli and gram-positive S. aureus for 24 h. A growth inhibition rate of 99.9% was observed for both bacterial samples. Thus, results suggested that addition of chitosan led to high antibacterial efficiency. The composite samples were also subjected to various water tests. Addition of chitosan to BC caused decrease in the swelling ratio. Water absorption capacity was found to be 97%. Water evaporation rates were also found to be higher as it took 48 h for water to be completely evaporated. After the analysis it was suggested that BC-Ch composites were able to maintain a suitable moisturized environment for wounds. On the concluding note, moist environment supports penetration of active substances, protects wounds against further bacterial infections and provides painless removal of wound surface after recovery. Hence, an ideal wound dressing should be able to maintain a proper moist environment for wound healing. BC-Ch composites incorporated these properties successfully, thus, they can be effectively used for wound dressing materials. Potential of bacterial cellulose-silver nanoparticles (BC-Ag) nanocomposites for biomedical applications was extensively explored by Shao et al. (2015) through various characterization techniques. The surface morphology was studied



CHAPTER 4 Characteristics and characterization techniques

using SEM micrographs. It represented a denser network for BC-Ag nanocomposites where AgNPs were present in the form of white spots. EDS analysis also confirmed existence of silver in BC matrix. FTIR spectroscopy for BC-Ag nanocomposites suggested that peaks shifted to a lower value indicating presence of strong interactions between OH groups of BC and AgNPs. Optical properties of nanocomposites were assessed through UV-VIS spectroscopy. Absorption at 414 nm was observed attributed to surface plasmon resonance of metallic AgNPs. This also confirmed the existence of silver in nanocomposites. Thermal properties resulted in two significant weight losses for BC-Ag nanocomposites. It was found that Tmax decreased from 363.2 C for BC to 256.6 C 286.4 C for BC-Ag nanocomposites. Reason for this was attributed to catalysis of CO2 elimination and acceleration in degradation process. XRD patterns of BC-Ag nanocomposites resulted in four characteristic peaks at 2θ values of 38.1, 44.3, 64.4, and 78.0 degrees attributed to (111), (200), (220), and (311) planes of metallic AgNPs introduced inside BC. Antibacterial activity for nanocomposites were studied at 37 C after contact time of 1 h. It was tested against gram-negative bacteria namely E. coli and Gram-positive bacteria namely S. aureus, B. subtilis, and C. albicans. After the analysis, it was found that BC-Ag0.05 nanocomposites were able to reduce E. coli, S. aureus, B. subtilis, and Candida albicans by 98.36%, 99.98%, 100%, and 89.6%, respectively. These results indicated exceptional antibacterial properties for BC-Ag nanocomposites. Hence, it was concluded that BCAg nanocomposites could serve as good antibacterial wound dressing materials such as bandages. Shao, Liu, Wu, et al. (2016) employed bacterial cellulose-silver sulfadiazine composites (BC-AgSD) for assessing its applications in biomedical field. Surface morphology was investigated using SEM micrographs which suggested a 3-D structure with ribbon shaped microfibrils network of BC matrix while BC-AgSD composites were presented as white spots on a denser network structure. Increasing the amount of AgSD caused the surfaces to become smoother and more compacted. FTIR spectroscopy revealed numerous characteristic peaks for BC, AgSD, and BC-AgSD composites as shown in Table 4.2. In case of BCAgSD composites, the peak intensities of AgSD were found to be increasing with an increase in AgSD loadings. A significant peak was observed at 3391/cm corresponding to free N-H. XRD analysis is used to investigate crystalline properties of materials. The XRD pattern of BC-AgSD exhibited six different peaks at 2θ values of 8.6, 10.01, 14.46, 16.62, 18.32, and 22.66 degrees which indicated that AgSD was successfully incorporated inside BC matrix. It was observed that different AgSD loadings caused the peaks of BC to become weak but were still evident indicating meaningful formation of composite. Thermal properties of composite revealed two significant weight loss stages. It was found that temperature decreased from 369.0 C for BC to 353.8, 348.7, 345.6, 340.0, and 331.4 C for BC1, BC2, BC3, BC4, and BC5, respectively. Cytotoxicity studies were performed to investigate the effect of AgSD in the BC matrix on proliferation of HEK293 cell line. It was observed that all materials possessed negligible toxicity

4.2 Biomedical applications of bacterial cellulose

and decrease in cell viability with increase in AgSD loadings. Also, AgSD caused no proliferation of HEK293 cells even at elevated concentrations. Antibacterial activity of composites was also analyzed. It was found that increasing the amount of AgSD loading caused rapid increase in the inhibition zones and after some time became stable. BC5 was found to be possessing best antibacterial activity with inhibition diameters of S. aureus and C. albicans to be 17.3 and 18.6 mm, respectively. It was concluded that BC-AgSD possess excellent antibacterial properties, thus, can be effectively used for applications in wound dressing. Potential of tetracycline hydrochloride loaded bacterial cellulose composite membranes (BC-TCH) was examined by Shao, Liu, Wang, et al. (2016) for its applications in wound dressing purposes. SEM, FTIR, antibacterial analysis were some of the characterization techniques applied to the composite membranes. SEM images revealed a 3-D structure with ribbon shaped microfibrils network of BC matrix while TCH particles were presented as white spots on the composite membranes. FTIR spectroscopy revealed various characteristic peaks as listed in Table 4.2. Studies of TCH release profiles from BC-TCH composite membranes suggested that increasing the concentration of TCH caused an increase in the TCH release profiles. Antibacterial activity of composites was also analyzed. It was found that increasing the amount of TCH loading caused rapid increase in the inhibition zones and after some time became stable. BC0.5 was found to be possessing best antibacterial activity with inhibition diameters of E. coli, S. aureus, B. subtilis, and C. albicans to be 45.7, 38.5, 34, and 12.1 mm, respectively. Thus, BC0.5 composite membranes represented excellent antibacterial growth activity and reduced the growth of E. coli, S. aureus, B. subtilis, and C. albicans by 99.98%, 100%, 100%, and 99.99%, respectively. Hence, it can be concluded that BC-TCH composite membranes have great antibacterial properties, thus, having a great potential in reducing infection and inflammation. Lucyszyn et al. (2016) discovered the usage of reconstituted bacterial cellulose films (RBC) as wound dressing materials. For this, various characterizations were applied to RBC films such as SEM, cytotoxicity, mechanical properties, etc. SEM micrographs were employed to assess the morphological changes taking place in the biocomposite films. A network type structure was displayed by SEM images. RBC was presented in the form of fibers of both macro scale and micro scale. Static contact angle (SCA) measurements were done to analyze behavior of RBC films in different liquid conditions. It was found that RBC did not possess high hydrophobicity which can be explained through low stability in water and NaCl solution. Addition of AG and GHXG led to increase in hydrophobicity of RBC films. XRD analysis of RBC films resulted in peaks at 2θ angle such as 14.4 (100), 16.8 (010), and 22.8 (110), corresponding to cellulosic crystallographic planes. RBC CrI value was found to be 75.9%. Inclusion of AG and GHXG resulted in no peak shifts. Mechanical properties were vastly studied for RBC films and RBC films introduced with hydrocolloids. It was observed that introduction of hydrocolloids (AG and GHXG) to RBC films caused an increase in the mechanical properties of RBC due to polysaccharide adhesion effect. Addition of



CHAPTER 4 Characteristics and characterization techniques

AG/GHXG to RBC caused 20% difference in Young’s Modulus and Strain at break. Fifty percent difference was observed by addition of AG and GHXG separately to RBC films for tensile strength. Cytotoxicity studies revealed interesting results where L929 cells were used for analysis after an incubation period of 72 h. It was observed that AG caused a decrease in cell viability by 30% and 23% at 3300 and 1667 μg/mL, respectively. Also, GHXG decreased cell viability by 16% and 9% at 3300 and 1667 μg/mL, respectively for L929 cells. Cell proliferation assay suggested that overall cell viability increased by 83% for AG/GHXG20 films as compared to GHXG20 and it increased by 88% for AG/GHXG40 films as compared to GHXG40. It was suggested that L929 cell proliferation was due to increased hydrophobicity, decreased porosity, and increased surface heterogeneity. Thus, it was concluded that RBC have a great potential as wound dressing material. Bacterial cellulose-tannic acid-MgCl2 (BC-TA-Mg) composites were successfully utilized by Zhang et al. (2020) for its applications in wound dressing. The composites were characterized using various techniques such as SEM, FTIR, XPS, cytotoxicity, antibacterial studies, etc. SEM results revealed that the BC matrix presented a porous network structure. Tannic acid and MgCl2 particles were attached to BC matrix in the form of small white particles. FTIR spectra revealed several characteristic peaks. This is shown in Table 4.2. All these peaks were also found in the FTIR spectra of BC-TA-Mg composites confirming successful introduction of TA onto BC matrix. The XPS analysis suggested that electrons were transferred from Mg21 to BC and TA. This confirmed chelation of BC, TA, and Mg21. Cytocompatibility of BC-TA-Mg composites revealed an increment in the cell viability as a result of increased concentration of Mg21. The highest cell viability was found to be 77.65 6 2.01% for the composite BC-TA6Mg. The reason was attributed to the fact that increase in concentration of Mg21 resulted in the chelation of BC, TA, and Mg21. Antibacterial activity for these composites was found to be larger in case of Gram-positive bacteria as compared to Gram-negative bacteria. Best antibacterial activity was observed for the composite BC-TA with a diameter of inhibition zone to be 17.30 6 0.53 mm against S. aureus, 15.07 6 1.05 mm against P. aeruginosa, and 14.39 6 0.80 mm against E. coli. A slight decrease in the antibacterial activity was observed on increasing the concentration of Mg21. Wang et al. (2020) explored the effects of BC functionalized with silk sericin (SS) and hyaluronic acid (HA) on wound dressing applications in the form of in situ as well as ex-situ modified BC-HA/SS composites. Different characterization techniques were applied to the composites such as FTIR, SEM, TGA, XRD, etc. FTIR analysis was used to describe various characteristic peaks for BC, SS, HA, and BC-HA/SS composite. This is presented in Table 4.2. In the case of composite samples, it displayed all the characteristic peaks of BC, SS, and HA indicating successful agglomeration of BC onto SS and HA. In addition, with this, a sharp peak at 1043/cm was found in ex-situ modified BC-HA/SS composite referring to enhancement in hydrogen bonding. SEM micrographs were used to determine the

4.3 Conclusion

morphology of composites. In situ modified BC-HA/SS composites displayed thin interwoven mesh fibrils of about 30 nm width while ex-situ modified BC-HA/SS composites displayed wider interwoven mesh fibrils of 50 100 nm. Surface morphology was also studied using AFM analysis. In situ modified BC-HA/SS displayed a smoother surface with 23.20 nm roughness resulting in more hydrophilic surface. On the other hand, ex-situ modified BC-HA/SS exhibited roughness of 30.38 nm. Crystalline structures of composites were described using XRD. Degrees of crystallinity were found to be 88.44% and 93.15% for in situ and exsitu modified BC-HA/SS composites, respectively. TGA analysis was used to determine the thermal stability of the composites. The first endothermic peaks were observed at 127.42 C and 134.67 C contributing 9.94% and 8.97% weight loss for in situ and ex-situ modified composites, respectively. Second endothermic peak was observed at 225 C contributing 11.38% and 6.12% weight loss for in situ and ex-situ modified composites, respectively. Endothermic peaks at 351.65 C and 406.53 C were observed which were attributed to thermal degradation of SS and depolymerization of glucose. These results suggested that HA was well agglomerated on in situ modified composite whereas SS was agglomerated well on ex-situ modified composite. Also, exothermic peaks were observed at 453.92 C and 479.18 C for in situ and ex-situ modified composites, respectively. Observations from TGA analysis suggested that ex-situ modified composite has much higher crystallinity and much better thermal stability as compared to in situ modified composites. No cytotoxicity was detected for the composites. Also, exsitu modified composites exhibited higher tensile strength and higher moisture content which was recorded to be 1.60 6 0.16 MPa and 79.06% 6 0.16%, respectively.

4.3 Conclusion Researchers have developed BC for myriad biomedical applications. This chapter discussed the implementation of BC in wound healing and dressing, diagnosis of ovarian cancer, shape memory material, preventing deterioration of salmon muscle, slowing down the lipid oxidation, lipase immobilization, tissue engineering, implantable devices in regenerative medicine, drug delivery, and bone healing. The morphology, microporous structure, microfibrillar, adhesion, 3-D structure, etc. of BC can be efficiently recognized with SEM/FESEM analysis. FTIR is capable to identify different characteristic peaks/groups present or impregnated in BC matrix. TEM and AFM characterizations were applied for determination of size, structure, and arrangements of fibrils within BC. TEM images have been used to confirm the results obtained from SEM images. XRD and TGA techniques have been used to study crystallographic planes and thermal stability of BC. DSC analysis is used to find out the glass transition temperature of BC while SCA measurements are done to analyze its behavior in different liquid conditions.



CHAPTER 4 Characteristics and characterization techniques

BET analysis is performed to determine the pore size, pore volume, and surface area of BC and its fabricated composites. Other characterization techniques associated with BC found in literature were XRF, 13C CP MAS NMR, and XPS. Major mechanical strength properties studied for BC identified were tensile strength, elongation at break, and Young’s modulus. Other properties investigated for BC were discovered to be antimicrobial activity, photocatalytic activity, hydrophilicity, water contact angle, surface tension value, physical and chemical stability, pore size, electrical conductivity, cell viability test, water absorption capacity, optical properties, cytotoxicity, cytocompatibility, etc. It can be concluded that getting acquainted with suitable characterization techniques for BC is very important for researchers to determine its apt biomedical applications. However, it is also important to consider the limitations of a particular characterization technique for BC.

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Trˇcek, J., & Barja, F. (2015). Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry. International Journal of Food Microbiology, 196, 137 144. Tsai, Y.-H., Yang, Y.-N., Ho, Y.-C., Tsai, M.-L., & Mi, F.-L. (2018). Drug release and antioxidant/antibacterial activities of silymarin-zein nanoparticle/bacterial cellulose nanofiber composite films. Carbohydrate Polymers, 180, 286 296. Ul-Islam, M., Khan, T., & Park, J. K. (2012a). Nanoreinforced bacterial cellulose montmorillonite composites for biomedical applications. Carbohydrate Polymers, 89, 1189 1197. Ul-Islam, M., Khan, T., & Park, J. K. (2012b). Water holding and release properties of bacterial cellulose obtained by in situ and ex situ modification. Carbohydrate Polymers, 88, 596 603. Ul-Islam, M., Subhan, F., Islam, S. U., Khan, S., Shah, N., Manan, S., et al. (2019). Development of three-dimensional bacterial cellulose/chitosan scaffolds: Analysis of cell-scaffold interaction for potential application in the diagnosis of ovarian cancer. International Journal of Biological Macromolecules, 137, 1050 1059. Vigentini, I., Fabrizio, V., Dellaca`, F., Rossi, S., Azario, I., Mondin, C., et al. (2019). Setup of bacterial cellulose production from the genus Komagataeibacter and its use in a gluten-free bakery product as a case study. Frontiers in Microbiology, 10, 1953. Wahid, F., Duan, Y.-X., Hu, X.-H., Chu, L.-Q., Jia, S.-R., Cui, J.-D., et al. (2019). A facile construction of bacterial cellulose/ZnO nanocomposite films and their photocatalytic and antibacterial properties. International Journal of Biological Macromolecules, 132, 692 700. Wang, X., Tang, J., Huang, J., & Hui, M. (2020). Production and characterization of bacterial cellulose membranes with hyaluronic acid and silk sericin. Colloids Surfaces B Biointerfaces, 195, 111273. Yin, N., Stilwell, M. D., Santos, T. M. A., Wang, H., & Weibel, D. B. (2015). Agarose particle-templated porous bacterial cellulose and its application in cartilage growth in vitro. Acta Biomaterialia, 12, 129 138. Yin, Q., Wang, D., Jia, H., Ji, Q., Wang, L., Li, G., et al. (2018). Water-induced modulus changes of bio-based uncured nanocomposite film based on natural rubber and bacterial cellulose nanocrystals. Industrial Crops and Products, 113, 240 248. Zhang, Z.-Y., Sun, Y., Zheng, Y.-D., He, W., Yang, Y.-Y., Xie, Y.-J., et al. (2020). A biocompatible bacterial cellulose/tannic acid composite with antibacterial and anti-biofilm activities for biomedical applications. Materials Science and Engineering C., 106, 110249. Zhijiang, C., Chengwei, H., & Guang, Y. (2012). Poly(3-hydroxubutyrate-co-4-hydroxubutyrate)/bacterial cellulose composite porous scaffold: Preparation, characterization and biocompatibility evaluation. Carbohydrate Polymers, 87, 1073 1080. Zimmermann, K. A., LeBlanc, J. M., Sheets, K. T., Fox, R. W., & Gatenholm, P. (2011). Biomimetic design of a bacterial cellulose/hydroxyapatite nanocomposite for bone healing applications. Materials Science and Engineering C., 31, 43 49.


Engineering scaffolds for tissue engineering and regenerative medicine


Ibrahim Fatih Cengiz1,2, Rui L. Reis1,2 and Joaquim Miguel Oliveira1,2 1

3B’s Research Group, I3Bs Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Cieˆncia e Tecnologia, Zona Industrial da Gandra, Barco, Guimara˜es, Portugal 2 ICVS/3B’s PT Government Associate Laboratory, Guimara˜es, Portugal

5.1 Introduction Orthopedic tissues are three-dimensional (3D) structures composed of cells and the extracellular matrix. Tissue grafts have been used with certain indications to treat lesions in the body. Given the challenges and shortcomings associated with both autografts and allografts (Pereira et al., 2019), the use of biomaterials has been proposed in addition to use biologics (Cengiz, Oliveira, et al., 2017; Cengiz, Oliveira, & Reis, 2018b; Cengiz, Pereira, Espregueira-Mendes, Reis, & Oliveira, 2019) and regenerative strategies (Cengiz, Silva-Correia, et al., 2017a; Cengiz, Oliveira, & Reis, 2014; Cengiz, Pereira, Espregueira-Mendes, Oliveira, & Reis, 2017; Pereira, Cengiz, et al., 2018). Engineered biomaterials, in particular scaffolds, have been changing the clinical practice for the treatment of lesions (Cengiz, Pereira, et al., 2018). Most of the regenerative strategies involve the use of scaffolds that are biomaterials processed into 3D structures to support and guide cellular activities to facilitate, guide, and modulate tissue regeneration via extracellular matrix synthesis of the cells. The ability of self-healing is considerable in vascularized tissues, while vascularization remains to be an outstanding challenge of tissue engineering targeting large defects. Different tissues have different characteristics regarding their matrix, cells, and biomechanics. Moreover, it is also known that tissues are heterogeneous in terms of the aforementioned features (Cengiz, SilvaCorreia, et al., 2017b; Cengiz, Pereira, Peˆgo, et al., 2017; Pereira et al., 2014; Pereira, Cengiz, Silva-Correia, Cucciarini, et al., 2016). Therefore engineering scaffolds is of critical importance since the outcomes of the application of the scaffolds depend on the scaffold itself and its interaction with the cells and body. While there is a range of scaffold producing and engineering methods with all their advantages and limitations, 3D-(bio)printing has impacted the field of tissue engineering more than other methods thanks to its ability to provide patient-specific Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 5 Tissue engineering and regenerative medicine

complex scaffolds in which biomaterial(s) with cells can be localized in 3D as designed. Herein, the relevant recent works on tissue engineering scaffolds that are proposed for meniscus, cartilage, and bone have been overviewed.

5.2 Scaffolds properties and characterization Scaffolds are 3D structures obtained via processed biomaterials that are engineered to support cell culture for regenerative applications to treat tissue lesions with certain features (Fig. 5.1) (Cengiz, Pereira, Silva-Correia, et al., 2017; Pereira et al., 2015; Pereira, Cengiz, Silva, Reis, & Oliveira, 2020; Pereira, Cengiz, Silva-Correia, Ripoll, et al., 2016). As an overall evaluation, it is a challenge to decide about the superiority of a biomaterial over others because the performance of the obtained scaffold depends on various factors associated with the engineering of the biomaterials and final applications. Fig. 5.2 illustrates the development of an implant through different levels of characterization and improvement to meet the needs before any trial in humans. Scaffolds can be categorized into several groups considering different aspects, such as origin (Reddy, Ponnamma, Choudhary, & Sadasivuni, 2021): naturally derived (Celikkin et al., 2017; Filippi, Born, Chaaban, & Scherberich, 2020) (including decellularized matrices (Taylor, Sampaio, Ferdous, Gobin, & Taite,

FIGURE 5.1 Overview of the major properties to be considered when designing a scaffold.

5.2 Scaffolds properties and characterization

FIGURE 5.2 Overview of the development of an implant through rigorous characterization and improvement to meet the preclinical needs.

2018)) or synthetic (Jenkins & Little, 2019); and composition: polymers (Jafari et al., 2017), ceramics (Ribas et al., 2019), metals (Tan, Tan, Chow, Tor, & Yeong, 2017), or composites (Turnbull et al., 2018). Commonly used natural polymers include but are not limited to collagen (Bahrami, Baheiraei, & Shahrezaee, 2021; Beketov et al., 2021; Kim & Kim, 2019), silk-based (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019; Ribeiro, Pina, Canadas, et al., 2019; Ribeiro, Pina, Costa, et al., 2019), gellan gum (Choi et al., 2020; Pereira, Silva-Correia, et al., 2018; Trucco et al., 2021), gelatin (Huang et al., 2021; Leucht, Volz, Rogal, Borchers, & Kluger, 2020; Sun et al., 2022), hyaluronic acid (Teng et al., 2021; Yan et al., 2020; Zhai et al., 2020), alginate (Sathish et al., 2022; Wulf et al., 2021; Zheng, Wang, Bai, Xiao, & Che, 2022), and chitosan (Li et al., 2021; Schmitt et al., 2021; Zuliani et al., 2021). Synthetic polymers include but are not limited to polycaprolactone (PCL) (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019; Dewey et al., 2021; Sani, Rezaei, Khoshfetrat, & Razzaghi, 2021), poly(lactic acid) (Ashwin et al., 2020; Patel, Dutta, Hexiu, Ganguly, & Lim, 2020; Tan et al., 2021), and poly(vinyl alcohol) (Fatahian, Mirjalili, Khajavi, Rahimi, & Nasirizadeh, 2020; Januariyasa, Ana, & Yusuf, 2020; Liu, Chen, Liu, Tian, & Liu, 2019). Bioactive glasses



CHAPTER 5 Tissue engineering and regenerative medicine

(Heras et al., 2020; Kang et al., 2020; Saatchi, Arani, Moghanian, & Mozafari, 2021), hydroxyapatite (Cao et al., 2020; Chen et al., 2021; Mondal et al., 2020), zirconia (Askari et al., 2020; Gaddam, Brazete, Neto, Nan, & Ferreira, 2021; Pereira, Cengiz, Maia, et al., 2020), and tricalcium phosphate (Duan, Ma, Song, Li, & Qian, 2021; Liu, Chen, Chen, & Zeng, 2021; Zhu et al., 2021) are examples of the widely used ceramic biomaterials; while titanium-based scaffolds (Huang, Pan, & Qiu, 2022; Pereira, Cengiz, Maia, et al., 2020; Yang et al., 2021) are often studied metallic scaffolds which can also be 3D-printed. Table 5.1 presents selected examples of commercial products for cartilage lesions. Depending on the Table 5.1 Examples of commercial products for the treatment of cartilage lesions. Product


Biomaterial 1 cells



BioTissue Technologies (Freiburg, Germany) Co.don (Teltow, Germany)

Polylactin/ polydiaxanon/ fibrin 1 autologous chondrocytes No scaffold 1 autologous chondrocytes

Kreuz et al. (2011), Ossendorf et al. (2007), Zeifang et al. (2010)

Arthro Kinetics Biotechnology (Krems, Austria)

Murine (rat tail) type-I collagen hydrogel 1 autologous chondrocytes Fibrin/hyaluronic acid 1 autologous chondrocytes Agarose/alginate hydrogel 1 autologous chondrocytes Bovine type-I collagen 1 autologous chondrocytes

Chondrosphere (ACT3D-CS/ ARTHROCELL 3D) CaReS-1S

Biocart II



RevaFlex (DeNovo ET) Novocart 3D

Histogenics (Waltham, Massachusetts) Tissue Bank of France (Lyon, France) Histogenics (Waltham, Massachusetts)

ISTO Technologies (St. Louis, Missouri) TETEC Tissue Engineering Technologies (Reutlingen, Germany)

Becher et al. (2017), Fickert et al. (2012), Siebold, Suezer, Schmitt, Trattnig, and Essig (2018) Petri et al. (2013), Schneider et al. (2011)

Eshed et al. (2012), Nehrer, Chiari, Domayer, Barkay, and Yayon (2008) Selmi et al. (2008)

Anderson et al. (2017), Crawford, DeBerardino, and Williams (2012), Crawford, Heveran, Dilworth Cannon, Foo, and Potter (2009) McCormick et al. (2013)

No scaffold 1 allogeneic juvenile chondrocytes Bovine type-I collagen/ Niethammer et al. (2014, chondroitin 2017), Zak et al. (2014) sulfate 1 autologous chondrocytes

Source: Reproduced from Cengiz, I. F., Pereira, H., de Girolamo, L., Cucchiarini, M., EspregueiraMendes, J., Reis, R. L., & Oliveira, J. M. (2018). Orthopaedic regenerative tissue engineering en route to the holy grail: Disequilibrium between the demand and the supply in the operating room. Journal of Experimental Orthopaedics, 5(1), 1 14 with permission. Copyright © 2018 Cengiz et al., licensed under CC BY-NC 4.0 (

5.2 Scaffolds properties and characterization

application on the target tissue, the choice of naturality, source, and composition are considered, since all biomaterials come with advantages and disadvantages. Typically, naturally derived scaffolds may provide a relatively higher level of bioactivity where cellular activities are supported relatively superior [e.g., silk fibroin (Sun, Gregory, Tomeh, & Zhao, 2021)] while synthetic scaffolds [e.g., PCL (Siddiqui, Asawa, Birru, Baadhe, & Rao, 2018)] have high reproducibility, customizable mechanical strength, and biodegradation while having relatively lower bioactivity. Depending on the processing methods, natural biomaterials may have inferior mechanical properties, or variation between batches, while the existing literature shows that via engineering, some known shortcomings could be overcome and superior scaffolds can be obtained such as scaffolds with increased suturability (Cengiz et al., 2020; Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019) and room-temperature gelation (Silva-Correia et al., 2013). Since the extracellular matrix of the tissue is synthesized by the cells, in the tissue engineering strategy the cells that can synthesize the specific matrix are used together with the scaffolds, where the scaffolds have certain roles. Both in vitro and in vivo (Fig. 5.3; Ricci et al., 2021), the function of cells (for

FIGURE 5.3 In vivo application of a multilayer collagen-hydroxyapatite scaffold. (A) An osteochondral lesion in the medial femoral condyle, (B) preparation of the implant site—9 mm deep regular box shape, (C) templating, (D) scaffold sizing, (E) press-fit implantation, and (F) bleeding from the subchondral bone. Reproduced from Ricci, M., Tradati, D., Maione, A., Uboldi, F. M., Usellini, E., & Berruto, M. (2021). Cellfree osteochondral scaffolds provide a substantial clinical benefit in the treatment of osteochondral defects at a minimum follow-up of 5 years. Journal of Experimental Orthopaedics, 8(1), 1 11, with permission. Copyright© 2020 Ricci et al., licensed under CC BY-NC 4.0 (



CHAPTER 5 Tissue engineering and regenerative medicine

instance, the properties of the synthesized extracellular matrix) depends on the features of the scaffold since cells sense the characteristics of the scaffold (Li, Xiao, & Liu, 2017; Zajac & Discher, 2008; Zonderland & Moroni, 2021), including the mechanical properties, chemical properties, surface properties, and microstructure (Cengiz, Oliveira, & Reis, 2018a), which may facilitate in an in vivo application the creation of a microenvironment through recruited endogenous stem cells’ paracrine activity (Caplan, 2007; Gunawardena, Rahman, Abdullah, & Abu Kasim, 2019; Karp & Teo, 2009; Kusuma, Carthew, Lim, & Frith, 2017; Zhou, Yamamoto, Xiao, & Ochiya, 2019), Given these, the challenges associated with scaffold features should be addressed via engineering.

5.3 Fabrication of scaffolds 5.3.1 Scaffold fabrication methods Several scaffold fabrication methods have been described in the literature, which were shown in Table 5.2. Each of these methods has pros and cons, and numerous biomaterials have been utilized as comprehensively reviewed by Collins et al. (2021), Koyyada and Orsu (2021), Cheng et al. (2019), Cidonio, Glinka, Dawson, and Oreffo (2019), and Sun et al. (2020). The scaffold fabrication method is selected considering the physical and chemical properties of the biomaterial to be used and the features of the obtained scaffold using that method. 3D-(bio)printing requires certain rheology of the biomaterial to be printed to make up the 3D structure, and using a patient-specific model, it is possible to produce scaffolds with correct size and shape, with control of positions of biomaterials and cells within the structure. On the other hand, conventional methods such as freeze-drying or solvent casting and particulate leaching are relatively less demanding methods, but do not provide directly a patient-specific scaffold unless using a patientspecific mold. Moreover, 3D-(bio)printing provides a larger degree of controllability of the microstructure than the conventional methods. Typically, the commercially available scaffolds are re-sized during the surgical intervention because the size/shape of the implant should match with the implantation site. The future strategies should address the current outstanding issues and limitations and the performance of novel tissue engineering scaffolds should be shown in clinical trials. 3D-(bio)printing methods (Table 5.3) stand out from the conventional methods by being able to provide scaffolds that are anatomically correct in terms of size and shape using a digital 3D model, heterogeneous in terms of structure and/or cellularity, and alive from the first moment since bioinks contain both scaffolding biomaterial and cells. The selection of scaffolding biomaterial and the manufacturing method are of critical importance to fulfill several needs that are associated with the application for the targeted tissue because not all biomaterials/fabrication methods can meet the requirements. Based on the targeted tissue, a clinically relevant scaffold should be suitable in

5.3 Fabrication of scaffolds

Table 5.2 Overview of scaffold fabrication methods and the principles. Scaffold fabrication method 3D-(bio)printing




Gas foaming

Main principle

Main feature


Layer-by-layer fabrication of scaffold alone (3D-printing), or with cells (bioprinting) based on a digital model of the scaffold via methods that are based on (including but not limited to) extrusion, droplet, or laser. It should be noted that there are also other methods under the rapid prototyping umbrella. Similar principle as 3D(bio)printing with the critical difference of an additional dimension of time, meaning that upon a stimulus, the morphology/function of the printed constructs can change over time. A porous structure is obtained via sublimation of the solvent

A 3D structure that is patient-specific in terms of size and shape with control of the positioning of biomaterial and cells.

Cengiz et al. (2020), Liu, Peng, et al. (2021), Zhang, Eyisoylu, Qin, Rubert, and Müller (2021)

A 3D-printed scaffold with the ability to alter a feature in a stimulusdepended manner.

Kim et al. (2020), Miao, Zhu, Castro, Leng, and Zhang (2016), Wang, Yue, et al. (2020)

Interconnected porosity with a range of pore sizes typically adequate for cell culture. Fibrous structures in a nano/micro scale.

Grenier et al. (2019), Safari et al. (2022), Zhang, Zhou, et al. (2019)

A fibrous structure is obtained by the collected fibers around a rotating collector, where the fibers were obtained via electrostatic forces from an electrically charged polymer. Solvent-free way of Porosity within the obtaining 3D porous foaming agent structures. (typically sodium bicarbonate) containing polymer is obtained due to the bubbles occurring as a result of escaping the gas (typically carbon dioxide).

Islam, Laing, Wilson, McConnell, and Ali (2022), Ma et al. (2021), Samadian, Khastar, Ehterami, and Salehi (2021)

Dattola et al. (2019), Kurakula and Koteswara Rao (2020), Manavitehrani et al. (2019)




CHAPTER 5 Tissue engineering and regenerative medicine

Table 5.2 Overview of scaffold fabrication methods and the principles. Continued Scaffold fabrication method Solvent casting and particulate leaching

Main principle

Main feature


A porous structure is obtained via leaching away of particles (typically salt) in a suitable liquid (typically water) after solvent evaporation.

The mold used for casting indicates the shape and size of the scaffold, while the microstructure could be altered by altering the size of the used particle. Modulation of the 3D porous structure via control of process parameters related to polymer, solvent, and temperature.

Sabzi, Abbasi, and Ghaleh (2020), Shalchy, Lovell, and Bhaskar (2020), Xie et al. (2021)

Phase separation

A polymer-rich phase is obtained via thermally induced phase separation, and then a porous structure is obtained by solvent removal. Decellularization A porous matrix is obtained via removal of cells from natural tissues.

Chen et al. (2020), Samadian et al. (2020), Wang, Kang, et al. (2020)

The extracellular matrix Goldberg-Bockhorn of the native tissue is et al. (2022), Lee, preserved. Olmer, Baek, D’Lima, and Lotz (2018), Wu et al. (2021)

Table 5.3 Advantages and limitations of 3D-(bio)printing techniques (Koyyada & Orsu, 2021; Sun et al., 2020; Zhang, Yang, Johnson, & Jia, 2019; Zheng et al., 2019). Advantages

Challenges and limitations




Availability of commercial printers More popular Availability of suitable biomaterials Multiple biomaterials can be printed Not suitable for lowviscosity biomaterials Lower precision Shear stress may negatively influence the cells

More economic printers Can work with lowviscosity biomaterials Fast

More precise Nozzle-free

Limited range of biomaterials Low cell density Cells may be damaged due to heat Clogging may occur in the nozzle

Expensive system Slower process Low capability to build up in 3D

5.3 Fabrication of scaffolds

terms of volume/size and shape, have required mechanical properties including suturability for certain tissues, have suitable biodegradation profile, provide the required amount of vascularity, have a suitable macro/micro/nano-structure and topography, allow native-like neotissue formation over time, and integrate sufficiently with the surrounding tissues.

5.3.2 Patient-specific scaffolds Patient-specific scaffolds (Cengiz et al., 2016; Cengiz, Pereira, Pitikakis, et al., 2017; Oner et al., 2017) have been recently available thanks to the progress on 3D-printers, bioinks, and software allowing the development of 3D models from clinical imaging modalities (Fig. 5.4). Moreover, patient-specific cells can be used either by seeded onto the 3D-printed scaffold or incorporated into the bioink that has a balance between polymer content and cell viability, based on the pressure to be used in extrusion-based bioprinting (Fig. 5.5; Cidonio et al., 2019). In addition to the need for scaffolds to be patient-specific, there are other requirements that should be met including mechanical properties, for example shape memory and suture retention strength, controlling infiltration, and vascularity through new blood vessel formation via controlling microstructure and the 3D design of the scaffold. Cengiz et al. (2020) proposed a novel scaffold system that

FIGURE 5.4 Overview of the development of patient-specific implants.



CHAPTER 5 Tissue engineering and regenerative medicine

FIGURE 5.5 The paradigm of cell printing using a bioink. (A) The bioink comprising a biomaterial with encapsulated cells that were cultured in vitro. (B) 3D diagram illustrating the balance between the polymer content, cell viability, and pressure that is of critical importance in bioprinting and biomechanical and biological performance of the construct. A (top) and B (bottom) were reproduced (with a minimal adaptation that involves the addition of letters [“A” and “B”] at the right-bottom corner of each figure) from Cidonio, G., Glinka, M., Dawson, J., & Oreffo, R. (2019). The cell in the ink: Improving biofabrication by printing stem cells for skeletal regenerative medicine. Biomaterials, 209, 10 24, with permission. Copyright © 2019 Cidonio et al., licensed under CC BY-NC 4.0 (

5.3 Fabrication of scaffolds

is called entrapped-in-cage where silk fibroin is entrapped in a PCL cage that is 3D-printed mainly utilizing the mechanical properties of PCL and biological properties of silk fibroin. Also, silk fibroin scaffolds that were reinforced with 3D-printed PCL mesh had an increased suturability, while allowing tissue infiltration and formation of new blood vessels in the scaffold (Cengiz, Pereira, Espregueira-Mendes, Kwon, et al., 2019). The process of fabricating patientspecific tissue engineering constructs can be organized into three major steps from the engineering point of view as follows: 1. Acquisition of medical image dataset related with the lesion that is aimed to be treated with a tissue-engineered scaffold. From the available clinical imaging modalities, magnetic resonance imaging (MRI) has been frequently used during the normal course of medical examination. Technically, computed tomography (CT) images can also work, but for the patient, MRI is safer than CT. However, to produce patient-specific implants, sufficient resolution and a 3D isotropic sequence imaging covering the entire defect that will provide a dataset should be achieved. The isotropic sequence indicates that medical images have the same resolution in all planes. For orthopedic tissues, static medical imaging can be used. Depending on the targeted tissue, such as bone, meniscus, or intervertebral disc (IVD), either T1- or T2-weighted MRI can be selected; the critical point is to distinguish/segment the targeted volume of interest from the rest of the volume. Thus a specific imaging protocol must be developed that will serve later for the creation of the 3D digital model. While other image file types could be used, a Digital Imaging and Communication in Medicine dataset would be more standardized with associated metadata. Duration of the MRI acquisition is also another parameter that should be considered. An acquisition for the 3D model creation needs a much longer time than a typical routine image and a different protocol should be used in the MRI system. Another challenge is that the patient should remain still during the acquisition to obtain a correct image dataset. 2. Segmentation of region of interests and eventually the volume of interest is a step that can be manual, semiautomatic, or automatic, based on the available software and protocol of digital extraction for the targeted tissue. Using either semiautomatic or automatic segmentation, manual revisions may be needed to enhance the accuracy of the 3D model to be obtained. In the case that the image dataset is not ideal for the segmentation, either the image acquisition should be repeated or the artifacts should be cleaned if possible. Following the segmentation, the 3D model that would be used for the 3D-(bio)printing should be created. A surface rendering process can be using the segmented image dataset. The obtained raw model that is typically a triangle mesh can be further refined digitally through smoothing, closing of any minor holes or open structures if there are. Before printing the structure, the 3D model should be in a format recognized by the printer that will be used, that is, in stereolithography (.stl) that is a typical format used in 3D-(bio)printing.



CHAPTER 5 Tissue engineering and regenerative medicine

3. The settings and the parameters related to the 3D-(bio)printing should be done considering the model and the biomaterial/bioink to be used, including but not limited to: a. the alignment of the model; b. need of any supporting structures for the printed model; c. the time between each layer; d. printing speed; e. layer thickness; f. if applicable: nozzle type, pressure, cell density, and crosslinking pathway; g. internal pattern (2D and 3D organization of biomaterial and/or cells); h. introduction of heterogeneity (or not) within the architecture of the structure; and i. sterility of the biomaterials/bioink and/or the final scaffold/construct. Following the consideration and optimization of the critical factors mentioned above, the 3D-(bio)printed constructs can be obtained, and depending on the strategy, the construct can be implanted as it is, or maturated in a typical incubator or in a bioreactor.

5.4 Conclusion Millions of people suffer from orthopedic lesions/disorders where one or more tissues are affected. While neither all lesions are symptomatic, nor all lesions should be surgically treated; moreover, upon a strong indication for the use of a scaffold, a single scaffold could not meet all the needs. Thus lesion-specific, patientspecific scaffolds are of critical importance. It has been recognized that controlling a wide range of properties within a scaffold would affect the cell behavior and the clinical outcome. 3D-(bio)printing has been changing the concept of scaffolds in tissue engineering, with the shift from biomaterial only scaffolds to living scaffolds via the use of bioinks. While the majority of the scaffolds being used currently in the clinics are produced conventionally, the future is patient-specific scaffolds that can be produced using 3D digital models and 3D-(bio)printing. The current outstanding issues are the mismatch/gap between the formed tissue and the native tissue in terms of biocomposition and biomechanics. Still, the use of scaffold depends on indications and contraindications defined for the lesion in question.

Acknowledgments The authors thank the financial support under the Norte2020 project (NORTE-08-5369FSE000044). The author I.F.C. thanks the TERM RES-Hub, Tissue Engineering and Regenerative Medicine Infrastructure project, funded by the Portuguese Foundation for


Science and Technology (FCT), and the funding through the project 2IQBIONEURO (ref. 0624_2IQBIONEURO_6_E). The FCT distinction attributed to I.F.C. under the Estı´mulo ao Emprego Cientı´fico program (2021.01969.CEECIND) is also greatly acknowledged.

Declaration of conflict of interest The authors declare that there are no conflicts of interest to declare.

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Koyyada, A., & Orsu, P. (2021). Recent advancements and associated challenges of scaffold fabrication techniques in tissue engineering applications. Regenerative Engineering and Translational Medicine, 7(2), 147 159. Kreuz, P. C., Mu¨ller, S., Freymann, U., Erggelet, C., Niemeyer, P., Kaps, C., & Hirschmu¨ller, A. (2011). Repair of focal cartilage defects with scaffold-assisted autologous chondrocyte grafts: Clinical and biomechanical results 48 months after transplantation. The American Journal of Sports Medicine, 39(8), 1697 1706. Kurakula, M., & Koteswara Rao, G. (2020). Moving polyvinyl pyrrolidone electrospun nanofibers and bioprinted scaffolds toward multidisciplinary biomedical applications. European Polymer Journal, 136, 109919. Kusuma, G. D., Carthew, J., Lim, R., & Frith, J. E. (2017). Effect of the microenvironment on mesenchymal stem cell paracrine signaling: Opportunities to engineer the therapeutic effect. Stem Cells and Development, 26(9), 617 631. Lee, K. I., Olmer, M., Baek, J., D’Lima, D. D., & Lotz, M. K. (2018). Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears. Acta Biomaterialia, 76, 126 134. Leucht, A., Volz, A.-C., Rogal, J., Borchers, K., & Kluger, P. (2020). Advanced gelatinbased vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents. Scientific Reports, 10(1), 1 15. Li, P., Fu, L., Liao, Z., Peng, Y., Ning, C., Gao, C., . . . Liu, S. (2021). Chitosan hydrogel/ 3D-printed poly (ε-caprolactone) hybrid scaffold containing synovial mesenchymal stem cells for cartilage regeneration based on tetrahedral framework nucleic acid recruitment. Biomaterials, 278, 121131. Li, Y., Xiao, Y., & Liu, C. (2017). The horizon of materiobiology: A perspective on material-guided cell behaviors and tissue engineering. Chemical Reviews, 117(5), 4376 4421. Liu, P., Chen, W., Liu, C., Tian, M., & Liu, P. (2019). A novel poly (vinyl alcohol)/poly (ethylene glycol) scaffold for tissue engineering with a unique bimodal open-celled structure fabricated using supercritical fluid foaming. Scientific Reports, 9(1), 1 12. Liu, S., Chen, J., Chen, T., & Zeng, Y. (2021). Fabrication of trabecular-like beta-tricalcium phosphate biomimetic scaffolds for bone tissue engineering. Ceramics International, 47(9), 13187 13198. Liu, Y., Peng, L., Li, L., Huang, C., Shi, K., Meng, X., . . . Cao, H. (2021). 3D-bioprinted BMSC-laden biomimetic multiphasic scaffolds for efficient repair of osteochondral defects in an osteoarthritic rat model. Biomaterials, 279, 121216. Ma, L., Yu, Y., Liu, H., Sun, W., Lin, Z., Liu, C., & Miao, L. (2021). Berberine-releasing electrospun scaffold induces osteogenic differentiation of DPSCs and accelerates bone repair. Scientific Reports, 11(1), 1 12. Manavitehrani, I., Le, T. Y., Daly, S., Wang, Y., Maitz, P. K., Schindeler, A., & Dehghani, F. (2019). Formation of porous biodegradable scaffolds based on poly(propylene carbonate) using gas foaming technology. Materials Science and Engineering: C, 96, 824 830. McCormick, F., Cole, B. J., Nwachukwu, B., Harris, J. D., Adkisson, H. D., IV, & Farr, J. (2013). Treatment of focal cartilage defects with a juvenile allogeneic 3-dimensional articular cartilage graft. Operative Techniques in Sports Medicine, 21(2), 95 99. Miao, S., Zhu, W., Castro, N. J., Leng, J., & Zhang, L. G. (2016). Four-dimensional printing hierarchy scaffolds with highly biocompatible smart polymers for tissue engineering applications. Tissue Engineering Part C: Methods, 22(10), 952 963.



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Recent trends in polymeric composites and blends for three-dimensional printing and bioprinting


Sriya Yeleswarapu, K.N. Vijayasankar, Shibu Chameettachal and Falguni Pati Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India

6.1 Introduction Three-dimensional (3D) printing has emerged as a powerful technique to fabricate objects of any size, shape, and complexity (Wang, Jiang, Zhou, Gou, & Hui, 2017). The versatility it offers with the materials or ease of fabrication made this technology extremely user-friendly in recent years. High throughput is another most attracting feature which allowed researchers to choose 3D printing over conventional fabrication methods. With its precise depositions, wide range of material usability, and complex patterning abilities it has become one of the pivotal fabrication techniques which can produce personalized products. However, since it is in its infancy, most of the objects that are being developed are still in investigation and therefore have not completely impregnated into clinical practice. To translate objects or scaffolds that are 3D printed, the initial criterion that must be considered is the material used for fabrication (Diment, Thompson, & Bergmann, 2017). Material plays a critical role in determining the functionality of the objects with regards to its interaction with the host (Diment et al., 2017). Considering this, a range of materials have been investigated such as metals, polymers, ceramics, fibers, etc. for their use in 3D printing of scaffolds or objects (Ahangar, Cooke, Weber, & Rosenzweig, 2019). However, the properties of each material are very distinct from one another, and all the ideal characteristics cannot be achieved with one single material. Hence, considering properties of the materials based on their application is quite necessary. For instance, material to fabricate extracorporeal objects such as prosthesis or orthosis should be mechanically stiffer to bear the loads and for materials as implants requires many other properties such as biodegradation, aiding tissue regeneration, promoting cellular attachment and proliferation at physiological conditions (Horst et al., 2010; Klute, Kallfelz, & Czerniecki, 2001). Lack of availability of materials with ideal

Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 6 Recent trends in polymeric composites and blends

properties led to the idea of developing composites which improves material characteristics in a synergistic manner. Although metals and ceramics are very prominent materials for many applications in biomedical field, lack of degradability and limited processability have forced researchers to explore the potential of polymers for similar applications (Ulery, Nair, & Laurencin, 2011). Polymers provide a wide scope for tuning of parameters by altering them physically or chemically (Liu & Ma, 2004). Also, the printability of polymers can be altered making them apt materials for biomedical applications using 3D printing. Though physical and chemical properties of polymers can be modulated as per need, properties that a polymer can impart to scaffold are often limited, thus demanding composite materials which can widen the range of tunability. It is due to these limitations, such as incomparable mechanical strength, discomfort, toxic degradation, etc., 3D printed scaffolds or objects are still not available to patients (Mason, Visintini, & Quay, 2016; Yang et al., 2018); hence, this led to the finding of many composite materials so as to translate 3D printing research. These modified polymeric composites have been gaining attention as they offer many advantages such as lightweight, high fatigue strength, corrosion resistance, durability, biocompatibility, and mechanical strength (Rajak, Pagar, Kumar, & Pruncu, 2019).

6.2 Need of synergistic approach in polymeric materials For materials to be used in biomedical applications, properties such as degradation, biocompatibility, biodegradation, and cell-favoring environment also have to be considered apart from mechanical features, although the latter is also key (Kim et al., 2011). Biomedical applications such as development of prostheses, orthoses, dental implants, bone substitutes, stent development, etc., are in major focus using 3D printing technology since most of the mentioned applications require personalization. Out of the mentioned products, prosthesis and orthosis come under extracorporeal objects while all the others have applications in vivo. As the area of application varies, it is quite critical to employ suitable materials for fabrication. For example, limb prostheses or foot orthoses that are externally fastened should be made of materials that are robust, corrosive resistant, water resistant, sustain heavy loads, bioinert, etc. On the contrary, materials for implants or bone substitutes have to be extremely biocompatible, cell-friendly, biodegradable, promote or allow tissue regeneration with reasonable mechanical stiffness. Moreover, the material should be printable, which implies they have to allow modification into many forms such as filaments, powders, or liquids suitable for 3D printing. Polymeric materials are widely used materials in the field of tissue engineering and regenerative medicine due to their availability, low melting points, high ductility, low price, and high flexibility. Moreover, polymers offer high degree of modification that can be modulated as per the application and is a major

6.3 Blends and composites of natural and synthetic polymers

advantage (Liu & Wang, 2020) Additionally, availability of polymeric inks in various forms makes them suitable for 3D printing applications. Many synthetic and natural polymers find their place developing products for biomedical research and applications (Gasperini, Mano, & Reis, 2014; Liu & Wang, 2020). However, objects that are developed with pure polymers are restricted for conceptual use and prototypes as they have inferior mechanical properties. With low mechanical properties they usually do not satisfy the functional characteristics that are required. The mechanical and structural strength of pure polymers can be extended only till a particular limit, which probably might not be sufficient for real-world biomedical applications. And hence, combining two or more polymers or adding other materials to polymers to form a composite is being investigated extensively to attain suitable characteristics by means of synergistic effect. The main aim of composites preparation is to modulate and enhance the material properties in terms of processability, printability, mechanics, stiffness, stability, and bioactivity (Guvendiren, Molde, Soares, & Kohn, 2016). Primarily 3D printing focused on fabricating objects with pure polymers but advances in technologies quickly lead to development of composite inks that are 3D printable. Biodegradable as well as nonbiodegradable polymers are available for 3D printing. However, most of the applications focus on using biodegradable polymers which may be synthetic or natural depending on their origin. Natural polymers provide a cell-friendly microenvironment to the encapsulated cells due to the presence of bioactive molecules. Cell-specific activities such as attachment, migration, proliferation, and differentiation (Gasperini et al., 2014) are often promoted by them. However, due to their inferior mechanical strength, 3D structures require quick crosslinking mechanisms (physical, chemical, or ionic crosslinking) to stabilize (Gasperini et al., 2014). Synthetic polymers are man-made polymers which are produced by modulating physical properties and chemical structures (Liu & Wang, 2020). The mechanical aspects can be modified as per requirement and mostly synthetic polymers tend to have superior mechanical strengths in contrast to natural polymers (Liu & Wang, 2020). However, due to the production process that involves use of harmful organic solvents, heat, and toxic substances, final polymers are mostly bio-inert and lack attachment sites for cell adhesion and other activities (Liu & Wang, 2020). Therefore composite derived from combining these natural and synthetic materials can be appreciated as they possess both characteristics.

6.3 Blends and composites of natural and synthetic polymers Synthetic polymers such as polylactic acid (PLA), polycaprolactone (PCL), polyurethane, acrylonitrile butadiene styrene (ABS) and poly D,L-lactic-co-glycolic acid (PLGA) and natural materials such as collagen, alginate, chitosan, silk, and



CHAPTER 6 Recent trends in polymeric composites and blends

gelatin are most used materials (Simionescu & Ivanov, 2015). Synthetic polymers lack bioactivity and natural polymers lack mechanical strength. Composites developed with combining these give a new set of characteristics features to the material which can be an advantage to the biomedical community. A summary of mostly used composites is mentioned in the next section with focus on the describing modulated parameters that have a positive impact when compared to the effect obtained by using material individually. The additive added for composite preparation may vary from ceramics, biomolecules, carbon fiber, natural, and synthetic polymers.

6.3.1 Synthetic polymers based composites Thermoplastics such as PLA, PCL, PLGA, and ABS are widely used synthetic polymers for fabrication of orthopedic, dental implants and prostheses applications due to their superior mechanical properties, easy processability, and huge availability (Liu & Wang, 2020). As modifying polymers to form filaments, powders, or viscous liquid is quite effortless, 3D printing techniques mostly prefer polymers. Moreover, it is due to their innate characteristic to liquefy at certain temperatures and solidify at lower temperatures depending upon their melting points, they are mostly preferred in fused deposition modeling (FDM) and extrusion-based printing techniques (Liu & Wang, 2020). PLA is an aliphatic biodegradable polyester which also possess high strength, high modulus, and is nontoxic and biocompatible in nature and is widely used in medical industry (Garlotta, 2001; Guvendiren et al., 2016). PLA can be made into filaments and can easily be molten at 200 C 230 C that makes it an excellent material for FDM 3D printing (Guvendiren et al., 2016). However, PLA undergoes thermal degradation at temperatures greater than 200 C which tends to affect the bulk properties of the scaffold. Also, it is brittle and has inferior compressive strength in comparison to bone. Ceramic additives such as calcium phosphate glass (Serra, Planell, & Navarro, 2013), hydroxyapatite (Senatov, Niaza, Stepashkin, & Kaloshkin, 2016), and natural polymers such as chitosan (Almeida et al., 2014) and alginate have a tendency to improvise pure PLA in terms of roughness, cell adhesion, hydrophilicity, mechanical and osteointegration properties. Inclusion of nutshells from walnuts, macadamia, and almonds to form a wood polymer composite enables its use as a material to fabricate lightweight prostheses and orthoses (Song, He, Han, & Qin, 2020). Similarly, PCL is another mostly used polymers that is biodegradable, chemically inert, and biocompatible with mediocre mechanical properties (Guarino, Gentile, Sorrentino, & Ambrosio, 2017; Guerra, Cano, Rabionet, Puig, & Ciurana, 2018). Also, the hydrophobic nature along with lack of binding sites do not promote cellular adhesion and proliferation which eventually leads to lack of regeneration with PCL alone (Liu & Wang, 2020). And hence, to impart biological properties to scaffolds, and to improve wettability, bioactive glass, hydroxyapatite, tricalcium phosphate, pristine graphene, alginate, and chitosan were added to PCL (Kundu, Shim, Jang, Kim, & Cho, 2015; Lee, Yu, Jang, & Kim, 2008; Li et al., 2014; Park, Lee, & Kim, 2011;

6.3 Blends and composites of natural and synthetic polymers

Shim et al., 2017). Furthermore, the degradability of PCL can also be modulated based on the molecular weight, due to which it is a material of choice for fabricating stents and drug delivery systems (Hollander et al., 2016). PLGA is another biodegradable polymer used to fabricate sutures as it has a property to undergo hydrolytic degradation (Gentile, Chiono, Carmagnola, & Hatton, 2014). It is a copolymer derived from lactic acid and glycolic acid which exhibits controlled degradation based on the ratio of monomeric units used to prepare the copolymer and thus has both hydrophilic and hydrophobic properties (Gentile et al., 2014). Despite biocompatible, it inhibits osteoconduction and hence pro-osteo materials such as ceramics and bioglass are mixed with PLGA to form a composite (Babilotte et al., 2021; Pan & Ding, 2012; Shuai, Yang, Peng, & Li, 2013; Yun et al., 2009). Inclusion of TiO2 into PLGA improves mechanical properties making it suitable for load-bearing applications (Rasoulianboroujeni et al., 2019). The scope of using ABS as a material for load-bearing application in biomedical applications is also enormous since the mechanical strength of ABS is high due to the presence of styrene units (Zuniga et al., 2015). Tensile strength of ABS can further be improved with addition of carbon fibers but improper optimization of additive might compromise toughness, ductility, and yield strength of plastic (Wang et al., 2019). And hence while preparing a composite, immense optimization and standardization is very critical to achieve the desired features and of course to achieve reproducibility.

6.3.2 Natural polymers based composites Natural biopolymers such as collagen, alginate, chitosan, silk, and gelatin are immensely explored as materials in biomedical and tissue engineering applications (Cui & Boland, 2009; England, Rajaram, Schreyer, & Chen, 2017; Hong et al., 2020; Shi et al., 2017; Stratesteffen et al., 2017; Yang et al., 2018; Yu, Zhang, Martin, & Ozbolat, 2013). The polymer network resembling native microenvironment provided by these biopolymers aids embedded cells to improve their functionality by nourishing them with appropriate biochemical cues (Guvendiren et al., 2016). Because of this, scaffolds developed with natural biopolymers create a positive impact on the overall functionality and regeneration of tissue in vivo. However, to use them as bioinks suitable for 3D printing, certain properties such as flowability, rapid gelation, shape fidelity, integrity, etc. have to be ascertained and evaluated. And hence, composites developed with natural polymers along with other materials are encouraged as natural polymers help maintain the cellfavorable properties while other stiff polymers or ceramics preserves the shape and mechanical stability along with providing the cells with biomechanical cues. Collagen constitutes the major portion of protein present in the extracellular matrix (ECM) of the tissue (Frantz, Stewart, & Weaver, 2010). Collagen is a natural choice of biomaterial because it is highly biocompatible and integrin receptors on cell membranes tend to recognize the RGD (tripeptide consists of Arginine, Glycine, and Aspartate) peptide domains on collagen, thereby



CHAPTER 6 Recent trends in polymeric composites and blends

enhancing cell adhesion and proliferation. Also, the viscosity of collagen is highly dependent primarily on its pH and temperature. Implying, at physiological pH and temperature of around 7.5 C and 37 C, collagen fibrils start to organize themselves to form a stable microstructure and thus collagen in liquid state forms crosslinked hydrogel. This interesting innate property of collagen makes it a bit inappropriate material for 3D bioprinting as the crosslinking is not so fast to develop a 3D stable structure or it is not so slow to flow out of nozzle. Often collagen starts to form gel with slight change in temperature due to which nozzle orifice is clogged and material does not come out. And since the viscosity of collagen is low in liquid state, encapsulated cells accumulate at the bottom during printing and patterning ability of 3D printer is lost. And hence mixing materials such as genipin which reacts with lysine groups of collagen, alginate which crosslinks with CaCl2, agarose, incorporation of nano-HA or synthetic polymer such as PCL is preferred to improve the mechanical properties and to ensure shape fidelity of the collagen-based scaffolds (Campos et al., 2019; Isaacson, Swioklo, & Connon, 2018; Yang et al., 2018). Gelatin, derived from collagen, is another prominent material used for 3D printed tissue engineering applications (GomezGuillen, Gimenez, Lopez-Caballero, & Montero, 2011). It is a thermoresponsive material that stays in solid form at low temperatures and forms liquid at higher temperatures whose behavior is quite contradictory to collagen (Gomez-Guillen et al., 2011). Stable constructs with shape integrity can be obtained with gelatin when printed at low temperatures, but once the printed scaffold reaches a temperature more than its melting point (usually near physiological temperatures) it turns into liquid compromising the shape and structure of the printed scaffold. Materials such as alginate, silk, oxidized nanocellulose, and methacyrlated hyaluronic acid (HA) helps gelatin-based scaffolds to be mechanically stable even at higher temperatures (Shubhra et al., 2010; Xu, Wang, Yan, Yao, & Ge, 2010). Chitosan, a chitin derivative biocompatible polysaccharide which also possess antibacterial, antifungal, analgesic, and hemostatic properties, because of its positive charge (Croisier & Jerome, 2013), lacks mechanical strength similar to other natural polymers. To impart mechanical strength to chitosan scaffolds, it is used in combination with either PCL, PLA calcium phosphate, gelatin, or alginate (Akkineni et al., 2015; Chen et al., 2014; Cheng & Chen, 2017; Dadhich et al., 2016; Gu, Tomaskovic-Crook, Wallace, & Crook, 2017; Wu, 2016). Silk is another prominent material which is known for its mechanical as well as biocompatible properties. Due to its ability to promote cellular activity it is mostly used as a material for fabricating bone, cartilage, ligaments, etc. Since the mechanical toughness and strength of silk are superior when compared to any other natural biomaterial, it is often used in combination with alginate, gelatin, collagen, or hyaluronic acid (Park et al., 2011; Singh, Bandyopadhyay, & Mandal, 2019; Wei et al., 2019). Also, the biological properties of silk are also extracted while using silk in combination with synthetic polymers. Synthetic polymers such as PLA and PCL have been used along with silk for fabrication of bone clips and bone tissue engineering (Vyas et al., 2021; Yeon et al., 2018). Very recently, the potential of

6.3 Blends and composites of natural and synthetic polymers

decellularized ECM (dECM) as a bioink is explored extensively. Since it is derived from tissue, though sufficient biochemical cues are provided to cells, the mechanical properties of material are inferior. Therefore improving the mechanical properties of dECM has high scope of research, and combining collagen, silk, alginate, and gelatin to form a dECM-based composite is being investigated (Gao et al., 2017; Gao, Park, Kim, Jang, & Cho, 2018; Hiller et al., 2018; Lee et al., 2018). When two materials are combined to form a composite, the disadvantages of one material are overcome by another and vice versa which turns out to be an advantage for the biomaterial research, as the composite is now more applicationfriendly. Fig. 6.1 shows few examples from literature illustrating 3D printed

FIGURE 6.1 Image illustrating 3D printed scaffolds printed with natural polymer-based composite along with live/dead stains showing biocompatibility and functionality of the scaffold. (A) 3D printed alginate/agarose scaffold with (B) live/dead of chondrocytes, (C) phalloidin-stained actin filaments (Yang et al., 2018), (D) 3D printed scaffold with silk fibroin/gelatin/ hyaluronic acid/tricalcium phosphate composite, (E) live/dead of adipose stem cells, (F) alkaline phosphatase (ALP) activity (Wei et al., 2019), (G) 3D printed alginate/collagen composite, (H) live/dead of chondrocytes, (I) phalloidin-stained actin filaments (Yang et al., 2018), (J) top view of a 3D printed scaffold, (K) viable primary Schwann cells encapsulated in the strand, (L) dorsal root ganglion (DRG) neurites aligned along 3D printed fibrin-factor XIII-HA strands (England et al., 2017), (M) 3D bioprinted corneal stroma equivalent, (N) live/dead staining keratocytes, (O) brightfield image of 3D bioprinted corneal structure (Isaacson et al., 2018), (P) silk gelatin based 3D printed scaffold with live/dead stain staining of chondrocytes, (Q) live/dead image of the cells (R) immunohistochemistry (IHC) staining for macrophages (Singh et al., 2019), (S) gelatin, alginate, fibrinogen based 3D scaffold, (T) CD31 1 stem cells, (U) CD31 1 cells along with Oil red O stain (Xu et al., 2010), (V) collagen decellularized extracellular matrix (dECM) and silk fibroin scaffold, (W) live/dead of osteoblasts, and (X) phalloidin-stained actin filaments (Lee et al., 2018).



CHAPTER 6 Recent trends in polymeric composites and blends

scaffolds fabricated with composites to achieve enhanced mechanical properties without compromising the cellular functionality.

6.4 3D printing techniques employed to print polymeric materials Fabrication of 3D object is possible with 3D printing technology which deposits material in a layer-by-layer strategy (Pati et al., 2014). Fast ongoing advancements in this field have enabled development of complex geometries with various materials. Due to the wide library of materials available which can be printed, this technology has also made its mark in biomedical and tissue engineering sectors. Although the concept of fabricating 3D structures is similar in every 3D printer, the printing technique and material being used, makes them applicationspecific. The various established 3D printing techniques that are available are (1) fused filament fabrication (FFF), also known as fused deposition modeling (FDM), (2) nozzle-based extrusion, (3) selective laser sintering (SLS), Vat-based polymerization techniques which include (4) stereolithography (SLA) and (5) digital light processing (DLP), and (6) laser-assisted bioprinting (LaBP) as shown in Fig. 6.2. Polymers or polymeric composites can be printed with these techniques, due to the flexibility offered by polymers to tune their parameters.

FIGURE 6.2 Illustrating various 3D printing techniques that are commonly used for printing polymeric composites characterized based on the form of starting material used during printing. (A and E) Filament-based extrusion-based 3D printing techniques, (B and F) powder-based 3D printing techniques, (C and G) liquid polymers based 3D printing techniques, and (D) laser-assisted bioprinting for hydrogel printing applications.

6.4 3D printing techniques employed to print polymeric materials

6.4.1 Extrusion-based 3D printing Extrusion is a process of expelling the material out through an orifice by applying a force. This is the same principle applied to a syringe to expel the material out of the barrel. Extrusion-based 3D printers are also based on the same principle with a control over material deposition which is in turn based on the design modeled in modeling software such as AutoCAD or solid works. However, the form of the material that is fed into the nozzle chamber slightly varies. There are two kinds of printers available which works on this basis, and they are FFF or FDM and 3D plotting. Fused deposition modeling FFF/FDM-based printers are widely used printers to fabricate 3D objects. It consists of a nozzle which is attached with a heating element. As the material in the form of filament approaches the orifice of the nozzle with the help of rotors, it melts due to the heat, gets extruded out of the nozzle, and is deposited on the platform (Zein, Hutmacher, Tan, & Teoh, 2002). During material deposition, subsequent layers are laid one above the other (Chia & Wu, 2015). Movement of nozzle and build platform may vary from manufacturer to manufacturer. Few printers have platform capable of moving in X-Y-direction while the nozzle moves only in Z-direction while few printers may have stable build platform and nozzle can move in all three XYZ-directions. Thermoplastics are ideal materials for FDM printing as they show a liquid-like behavior at temperature greater than melting point and almost immediately form solid when that temperature is decreased (Chia & Wu, 2015). Moreover, filament production with thermoplastics is quite straightforward. The material fed into the printer is in the form of filament and many synthetic thermoplastics such as ABS, PLA, PCL, and poly(propylene fumarate) (PPF) are common materials that are made into filaments and are used to print objects (Chiulan, Frone, Brandabur, & Panaitescu, 2018; Hutmacher et al., 2001; Zein et al., 2002). Materials choice can also be extended to composites made of PLA/ABS, graphene/PCL, PPF/ PCL, and PCL/PEG for FDM-based printing. Using these materials biomedical applications such as prosthetics and orthosis, bone implants were fabricated (Burn, Ta, & Gogola, 2016; Chiulan et al., 2018; Park et al., 2018; Placone & Engler, 2018; Teixeira, Aprile, Mendonca, Kelly, & da Silva, 2019; Zuniga et al., 2015). FFF technology has become the most common form of 3D printing technology due to its advantages such as low cost, ease of operation, wide range of material availability, and ability to deposit multiple materials. However, since the starting material is in the form of filament, composites developed should also be made into filaments while maintaining the material homogeneity. Moreover, since the processing temperatures of the material extrusion are very high, temperaturesensitive biological factors as well as viable cells cannot be incorporated into the material (Chia & Wu, 2015).



CHAPTER 6 Recent trends in polymeric composites and blends 3D plotting Extrusion-based printing technique deposits material in the form of continuous strands onto the substrate (Gu, Fu, Lin, & He, 2020). It is widely used technique for bioprinting because of its flexibility and affordability (Gu et al., 2020). This approach is also very popular due to the availability of wide range of materials that are compatible with this technique (Landers, Hu¨bner, Schmelzeisen, & Mu¨lhaupt, 2002). This technique basically works on the principle of expelling ink from the nozzle on application of mechanical or pneumatic forces. This extruded ink is deposited onto the substrate to form a desired architecture. Based on the technique that is utilized to drive the material out of the nozzle, this extrusionbased bioprinting is divided into three types, namely, pneumatic based, piston driven, and screw driven (Gu et al., 2020). Pneumatic based uses compressed air to push the material, while piston driven and screw driven use a mechanical force for ink ejection. Piston is connected to a guide screw which is in turn connected to a motor, to translate rotational motion of motor to linear motion, thereby pushing the material out of the nozzle (Gu et al., 2020). This technology is very versatile and finds applications with materials that have a wide range of viscosities. Also, it is a reliable technique to use in fabricating viable scaffolds in tissue engineering, as it is compatible with cells and various biomaterials (Landers et al., 2002). Many materials and composites such as collagen (He et al., 2018), gelatin (Wang et al., 2006), alginate (Fedorovich et al., 2012), gelatin/chitosan (Chang, Nam, & Sun, 2008; Ng, Yeong, & Naing, 2016), dECM/alginate (Gao et al., 2017), gelatin/fibrinogen (Xu et al., 2007), gelatin/alginate (Yan et al., 2005), collagen/chitosan (Ma et al., 2003), and nanocellulose/alginate (Nguyen et al., 2017), various dECM-based bioinks derived from many tissues such as adipose (Pati et al., 2014), heart (Pati et al., 2014), liver (Lee et al., 2017), cartilage (Pati et al., 2014), and skin (Kim et al., 2018) can be used with this technique. The usability of this technique in many applications can be enhanced with slight modifications to the nozzle, for instance coaxial nozzle design. However, the printed structure is highly dependent on the nozzle diameter, print speed, viscosity of bioink, force being applied, ink extrusion pressure, temperature, etc. (Gu et al., 2020). Additionally, the clogging of nozzle is said to be a major setback, yet this technique is widely used all over the world (Azad et al., 2020).

6.4.2 Vat polymerization This is an approach that uses the principle of photopolymerization of photosensitive materials on exposure to precisely controlled laser light. The two basic types of printing modalities that utilizes this approach are SLA and DLP. The basic setup in both the modes comprise of a transparent tank to hold resin (photosensitive material), photo-optics assembly, and platform to hold the printed structure (Quan et al., 2020). Typically, material is poured into the resin tank and based on the design, laser light is directed onto the resin. At places where laser light is showered in X-Y-direction, liquid material in that area solidifies forming one layer of the

6.4 3D printing techniques employed to print polymeric materials

printed structure on the platform (Wang, Goyanes, Gaisford, & Basit, 2016). Role of the platform is to move in Z-direction after one layer of printing is finished, making space for the uncrosslinked material to fill the gap and is ready for second layer polymerization (Melchels, Feijen, & Grijpma, 2010). Resolution of the printed structure is dependent on the laser spot size as well as on the movement of the platform in Z-direction. Other parameters such as laser power, scanning speed, laser wavelength, exposure time, and postprocessing affect the precision of printed structure in this technique (Melchels et al., 2010). The major difference between SLA and DLP is that SLA uses a pointed laser as light source, whereas DLP uses a divergent beam of light as source and hence a complete layer gets cured at one time in DLP technique (Quan et al., 2020). Because of light beam, the DLP technique is a much smoother and faster technique than SLA-based system in terms of construct fabrication time (Gu et al., 2020). The primary characteristic that a material should possess for it to be used for Vat-based 3D printing techniques is light sensitivity. Most of the available polymers are not light-sensitive, and hence, materials are modified to make them light-responsive. To behave as photosensitizers, materials such as gelatin, poly(ethylene glycol) (PEG), and silk were modified to form gelatin-methacryloyl (GelMA), poly(ethylene glycol diacrylate) (PEGDA), and silk-methacryloyl (SilMA) (Kim et al., 2018; Ma et al., 2016). Starch methacrylation was also explored to evaluate its potential as a material for DLP-based 3D printing strategy (Noe`, Tonda-Turo, Chiappone, Sangermano, & Hakkarainen, 2020). Recent literature reports the use of methacrylated chitosan as a material for vat polymerization. (Tonda-Turo et al. 2020). It is worth noticing that many materials that can undergo methacrylation can be used as potential materials for Vatbased 3D printing technologies. Materials such as PPF/diethyl fumarate (DEF), PPF/DEF-HA, PDLLA (Poly(D,L-Lactic Acid))/HA, and poly(ethylene glycol) dimethacrylate (PEG-DMA) (Lee et al., 2007; Luo, Fer, Dean, & Becker, 2019) were used with this technology for bone and cartilage tissue engineering (Bose, Vahabzadeh, & Bandyopadhyay, 2013; Cui & Boland, 2009). Even materials such as PLA and polyurethane diacrylate are also printed with SLA technique (Danilevicius et al., 2015; Petrochenko et al., 2015). SLA-based technology is popularly used to fabricate high-resolution, intricate, precise complex geometry due to the optical source and assembly contained with the printer. However, the starting material used in these techniques might not be cytocompatible, and hence, these are mostly used for scaffold fabrication which are further used to seed cells postprinting (Gu et al., 2020). Yet, because of possibility of modifying polymers, nowadays these modified polymers are widely explored as materials for DLP and SLA-based techniques from the conventional proprietary resins.

6.4.3 Powder bed fusion Powder bed fusion is a 3D printing technique that uses material in powder form unlike the other printing techniques which uses material in liquid form such as SLA, DLP, or 3D plotting. There are two different techniques that use material in



CHAPTER 6 Recent trends in polymeric composites and blends

powder form, and they are SLS and binder jetting. However, the technique with which powder material is fused together to form a 3D structure varies. Selective laser sintering SLS is a powder-based 3D printing technique which uses material in powder form. The principle of operation behind this technique is sintering of material when it is exposed to a high-power laser. Powdered material is rolled onto the base platform and a high-power laser beam is directed onto the material as per the CAD design. The particles in the area that is exposed to laser fuse and bind together forming the first layer of the 3D structure. Once the first layer is done, the platform lowers, and fresh unbound powdered material is rolled onto the surface. This process continues until the final layer of structure gets printed (Gu, Meiners, Wissenbach, & Poprawe, 2012). The 3D printed object is retained inside the powdered material and the unused material can be dusted to obtain the actual part. Unbound material that does not fuse together can be reused. Print finish depends on parameters such as laser power, scanning speed, and size of the particle of the powdered material (Gibson & Shi, 1997). The main advantages of this technique are it offers support-free fabrication, fabrication of high-resolution objects, and material availability. However, void formation due to improper fusion needs to be tackled to avoid print failures. Theoretically most of the commercially available polymers can be used for SLS in powder form; however, sintering modifies properties of polymers, which limits their use for SLS technology. PCL and polyamide are widely used polymers for SLS technique (Muzaffar et al., 2019). Literature reports that materials such as PLLA and PHA can also be used as materials for SLS technology (Chiulan et al., 2018). Binder jetting or powder liquid 3D printing Binder jetting or powder liquid 3D printing is also a similar to SLS technique but with a slight modification. A binder in liquid form is pumped and dropped on the powder layer according to the 3D design (Saroia et al., 2020). Binder allows particle agglomeration wherever it is selectively deposited to bind the particles together. As soon as the first layer is finished, platform is lowered down again to fill the surface with a fresh material ready to form second layer (Sachs, Cima, & Cornie, 1990; Saroia et al., 2020). The process continues till the last layer of the part is fused. Similar to SLS, printed object is retained in the unbound material and can be easily removed. Postprocessing of printed objects by using furnace cycles helps to improve the strength (Diegel, Withell, de Beer, Potgieter, & Noble, 2012). Complex structures with good print fidelity and stability can be obtained with the technique. Structures with variable mechanical strength can also be obtained with this technique by modulating the ratio of binder to powder (Gokuldoss, Kolla, & Eckert, 2017). Moreover, as sintering of particles is not involved as seen in SLS, and hence voids or crack formation due to residual stress is negligible. Various parameters such as binder viscosity, binder ejection rate, deposition speed, powder size, and more importantly the interaction of powder

6.5 Application of value-added polymers

and binder play a critical role in determining the success of the printed object (Gokuldoss et al., 2017). Nonetheless, parts fabricated with this technology have inferior mechanical strength and have coarse architecture when compared to parts fabricated with SLS. Materials such as PLLA, lactose, and methyl cellulose are made into powders, and cationic methacrylic ester copolymer, ammoniomethacrylic acid copolymer, and polyvinylpyrrolidone were used as binders developing tablets to release drugs (Deng et al., 2007; Rowe et al., 2000).

6.4.4 Laser-assisted bioprinting LaBP is a droplet-based, noncontact, nozzle-free technique to precisely deposit material along with cells onto the substrate (Gu et al., 2020). It basically consists of a laser pulse source, a ribbon with a coating of bioink and a receiving substrate (Jana & Lerman, 2015). The ribbon is made up of an energy-absorbing material often made up of gold or titanium, and is sandwiched between glass and bioink layer (Catros et al., 2011). The principle behind this 3D printing technique is that a laser source is directed onto the glass which is holding energy-absorbing ribbon and bioink. This ribbon absorbs the energy, and a high-pressure bubble is formed due to the local evaporation. Further this bubble pushes material containing cells onto the substrate (Gu et al., 2020; Guillemot, Souquet, Catros, & Guillotin, 2010). Laser assisted bioprinting (LAB) technique is very promising way to develop structures at high speed with high precision (Serra et al., 2006). Its ability to deliver single cell allows deposition of various cell types in spatial volume to replicate the complex coculture conditions in the native structure (Barron, Krizman, & Ringeisen, 2005; Guillemot et al., 2010). Furthermore, problems such as damage to substrate and cross-contamination are almost negligible due to its noncontact approach (Gu et al., 2020) and is said to be more suitable for in situ bioprinting.

6.5 Application of value-added polymers Biomaterials developed by polymeric composites act as a template to support cell attachment, migration, proliferation, and many other cellular activities due to the micro-morphology (O’Brien, 2011). Moreover, materials that comply with the tissue mechanical properties aid in rapid integration which helps in faster regeneration. The use of biomaterial composites with 3D printing techniques enhances the features of fabricated scaffolds. Structural features such as complex geometry, well-defined porosity, high resolution, defined cell positioning, structural similarity is imparted to the scaffolds. With these enhanced features, the cellular responses and degradation can be even more improved, thereby enhancing tissue integrity, and hence raising the chances of acceptance and regeneration in vivo (O’Brien, 2011). Many applications ranging from hard tissues such as scaffolds for dental implants, scaffolds for bone regeneration, to soft tissues like patches



CHAPTER 6 Recent trends in polymeric composites and blends

for skin regeneration along with antibacterial and wound healing abilities, extracorporeal devices such as prostheses and orthoses, and implantable devices such as stents are being fabricated with polymeric composite materials. Biocompatibility and biodegradability by actively promoting cell attachment, migration, proliferation, and eventually differentiation are considered as primary elements that an implant is supposed to possess. The differentiation of migrated cells on the implant allows cells to deposit their own ECM, thus helping in regeneration of the lost tissue. Also, the rate of degradability plays a key role so as to support the newly formed tissue. Due to cell-favorable properties, ceramics often find place in developing dental implants. However, due to few disadvantages of ceramics such as brittleness, inferior mechanical properties, and low fracture strength, they are used in combination with polymers (Miyazaki, Kawashita, & Ohtsuki, 2015). Properties such as improved cell migration, proliferation, and alkaline phosphate along with enhancement in scaffold’s surface roughness, porosity, and wettability can be imparted to PCL scaffold by combining it with β-tricalcium phosphate (β-TCP) (Park et al., 2017). Addition of TCP to polymers also facilitates early revascularization, good structural integrity, and better osteointegration which aids in speeding up the process of bone regeneration in vivo (Khojasteh et al., 2013; Li et al., 2014). Similarly, inclusion of carbon fiber to polyetheretherketone (PEEK) has led to the development of composite that has mechanical properties similar to cortical bone (Han et al., 2019). Hydroxyapatite is another promising material that is used along with polymers such as PLA and PCL as a composite to improve many properties such as tensile and flexural strengths, cell attachment and proliferation (Chen et al., 2019; Jiao et al., 2019; Mondal et al., 2020; Zhang et al., 2016, 2017). Altogether, composites developed from ceramics and polymers have enormous potential toward development of bone or dental implants to aid in faster regeneration of the damaged tissue, by promoting properties in synergistic manner. Prostheses and orthoses are other applications where polymeric composites play a crucial role in imparting critical properties to the products (Scholz et al., 2011). Prostheses are artificial assistive devices that assist a missing body part lost to trauma or injury or any disease condition to retrieve functionality. Similarly, orthoses are also artificial external assistive devices that are intended to support the limbs or spine or to correct the posture by preventing unnecessary movements. There are many different types of prostheses depending on the site of injury, namely, prostheses for lower limb, upper limb, shoulder, craniofacial, transradial, neck, foot, transtibial, transfemoral, etc., and orthoses for spine, arm, cervix, wrist, knee, hip, ankle, foot, etc. They must be designed and fabricated to cater the functional needs of the individual depending on the site of placement. Due to which few characteristics such as load bearing, high mechanical strength, and water resistant, lightweight is considered quite necessary. Since, a single material cannot provide all the necessary characteristics, often composites are considered for such applications. Most of the synthetic polymers intended for this do possess mechanical strength, but to enhance them composites such as ABS

6.6 Current challenges and possible solutions

reinforced with polycarbonate, carbon and Kevlar fibers into ABS, PLA, and nano carbon powder and polyurethane/PLA are being investigated (Jain & Tadesse, 2019; Tao, Shao, Li, & Shi, 2019; Wang et al., 2019). This concept of polymeric composites is also used for fabricating soft tissues such as skin. Combination of polymers such as chitosan/PLGA, alginate/PVA, collagen/PCL, and PCL/HA have been reported to promote many properties such as biocompatibility, neo tissue formation, improved cell responses, lightweight, and moisture retention (Dai, Williamson, Khammo, Adams, & Coombes, 2004; Shalumon et al., 2011; Wang et al., 2013). Functionalization of PVA can be improved with addition of natural material silk, as it promotes wound closure even in diabetic condition (Chouhan, Janani, Chakraborty, Nandi, & Mandal, 2018). The abovementioned examples illustrate that on addition of natural materials to synthetic material, bio favorable properties of synthetic polymers can be improved. On the other hand, the viscosity profile of silk that can be used for skin tissue engineering applications using 3D printing is enhanced by addition of PEG (Zheng et al., 2018). It is worth observing that many properties such as water uptake, printability, promoting cellular activity, and neo-vascularization can be imparted to materials by adding bioactive materials to synthetic polymers. Bacterial nano-cellulose (BNC) is a potential material that is used in fabrication of artificial blood vessels, but it possesses inferior mechanical properties. Hence, addition of PVA to BNC improves many mechanical properties such as compliance like an artery, water permeability, suture retention, and burst pressure which are very critical (Tang, Bao, Li, Chen, & Hong, 2015). The polymeric composite made of PLA and chitosan was found to be promoting cardiomyocyte activity and also enhanced production of sarcomeric α-actinin and troponin 1, thereby promoting myocardium regeneration (Liu et al., 2018). Another notable material that has been very popular in 3D bioprinting and tissue engineering area is dECM derived from specific tissue (Pati et al., 2014). But due to its weak mechanical properties it is often used in combination with other natural polymers. dECM obtained from vascular tissue in combination with alginate promoted proliferation, differentiation, and neovascularization, and also allowed printability of hydrogel (Curley et al., 2019).

6.6 Current challenges and possible solutions Pertaining to the biomedical applications such as extracorporeal objects, and implants or tissue engineered scaffolds, the existing 3D printable materials that are in research have few limitations. The primary concern for 3D printing is lack of availability of printable materials with appropriate physical properties. Material properties such as biocompatibility, biodegradability, or resorption, load bearing, and mechanically tough are very essential when a material is being considered; however, compatibility among materials is also key when we consider composites as material for fabrication (Fu, Feng, Lauke, & Mai, 2008). Hence, it is very



CHAPTER 6 Recent trends in polymeric composites and blends

crucial to select materials that can be easily combined with one another. For example, if surface property of one material is hydrophobic and the other is hydrophilic, there could be improper binding among materials and nonhomogenous composite would lead to formation of voids, or fiber pull outs, etc. The next challenge is that it is desirable to fabricate products that are light in weight for many applications, which can be attained by adding carbon fibers or fillers (Saroia et al., 2020). However, the extent of additives added should be optimized, because as the ratio of additives increases, primary mechanical properties of the base material might be compromised. Additionally, excessive incorporation of fillers might lead to nonhomogenous mixing, and the composites are prone to void formations, cluster formations by filler materials, and cracks (Mehdikhani, Gorbatikh, Verpoest, & Lomov, 2019). Any improper binding and lack of interaction between polymers in a composite might lead to voids and cracks which would diminish the mechanical properties drastically. Another crucial criterion is selection of optimal particle size of the additive to achieve homogenous distribution of the additive (Fu et al., 2008). This is also important when we use the composite as a starting material for extrusion-based 3D printing technique, since particle larger than nozzle diameter will clog the nozzle. Structural instability due to inferior mechanical properties is the major challenge that natural polymers pose while fabricating scaffolds or constructs. Therefore, viscosity of the material and its potential to crosslink after laid down is very critical and challenging. Most of the materials that encompass living cells often lack this property due to which they tend to spread as soon as they are laid down; hence fabricating 3D structures with natural materials alone is quite difficult. Apart from addressing challenges posed due to materials, there are few hurdles that are created by 3D printers too, which are inevitable. In FDM printers, the material undergoes instantaneous heating and immediate cooling leading to formation of voids inside the structure which is very prominent (Saroia et al., 2020). Similarly, in SLA or SLS technologies, there could be some binding variation in vertical direction versus horizontal direction as the layers get exposed to gradient of temperatures. All these parameters would contribute to the anisotropic properties of the scaffolds, and hence, postprocessing time and optimum printing conditions play a substantial role in determining the success rate of printed scaffold. The complexity furthermore increases, when 3D bioprinting has to be done, implying incorporation of living cells for developing in vitro tissues. All the material and printing parameters must be very well optimized and standardized to ascertain the viability of the cells in printed construct. The scope of developing newer composites to suit applications is very wide. Although many materials that are being used nowadays are biocompatible, developing a construct with material that has optimal degradation, nontoxic leaching substances, immunological response, etc., with suitable mechanical behavior is still underway. Due to the advancements, the flexibility of choosing material to develop composites for 3D printing is improving. And hence, it is worth speculating that in few years down the line, composites that are suitable for biomedical applications will come into translation.


6.7 Conclusion Though the currently available synthetic or natural polymers are 3D printable they may not match up with all the desired characteristics for many applications. While the biomedical applications that could be developed with 3D printing technology are enormously increasing and, their requirements could only be equaled up by utilizing various combinations of material composites. Many polymeric composites that can provide unique and distinct properties are being evaluated at laboratory and research level for fabricating products that are mechanically and functionally stable. To explore the potential of 3D printing technology, selection of materials plays a key role, and its optimization is inevitable. The idea of composite preparation widens the practical use of many materials to redefine their use for specific applications. Overall, it can be summarized that the concept of composites as material for 3D printing allows development of personalized products that are patient-specific with appropriate desired product characteristics.

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CHAPTER 6 Recent trends in polymeric composites and blends

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Polymers for additive manufacturing and 4D-printing for tissue regenerative applications


Bhuvaneshwaran Subramanian1, , Pratik Das2, , Shreya Biswas2, Arpita Roy3 and Piyali Basak2 1

School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India 2 School of Bio-Science and Engineering, Jadavpur University, Kolkata, West Bengal, India 3 Polymer Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology (ISM), Dhanbad, Jharkhand, India

7.1 Introduction Traditional methods of manufacturing an object involve subtractive approaches, that is, removal of material through milling, machining, carving, shaping, etc. In additive manufacturing (AM), objects are created by depositing layer upon layer using a hardware system guided by a computer-aided design (CAD) software. Each of these successive super-thin layers bonds to the partially melted preceding layer. Architectural files (in stl format) are created by “slicing” the model object into ultra-thin layers. This information is then used by the CAD to direct the path of a nozzle or print head for precisely depositing the layers in place. Particular areas are melted by laser or electron beams so that they join after cooling down to give the object its final predetermined shape (Junk & Kuen, 2016). Hideo Kodama pioneered the initiation of AM in the 1980s. Kodama, then working at the Nagoya Municipal Industrial Research Institute, Japan, published detailed protocol and information on the production of a solid model using AM (Gokhare, Raut, & Shinde, 2017). In 1987, AM first appeared coupling with stereolithography (SL) from 3D manufacturing systems. Stereolithography (SL) refers to the process of curing layers of UV light-sensitive polymers. In 1986, Charles Hull patented this stereolithography as the first rapid prototyping system, which reduced the manufacturing time considerably and was thus eventually commercialized. The first commercially available AM system was SLA-1 (stereolithography apparatus), which was later upgraded to the extensively popular version 

Contributed equally to the work.

Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 7 Polymers for additive manufacturing

SLA 250 model, which is capable of precisely manufacturing complex designs, even to the minutest details (Martinez, Basit, & Gaisford, 2018). New generation machines started appearing from the year 2000. “Quadra”, a 3D inkjet printer capable of depositing hardened photopolymer using 1536 nozzles and UV light source, was introduced by Objet Geometries of Israel. Later on, Objet upgraded its “Quadra,” which could make polycarbonate, acrylonitrile butadiene styrene (ABS), and polyphenylsulfone parts. “Prodigy” was marketed by Stratasys, which ABS could manufacture plastic parts. T612 systems were introduced by Solidscape in 2013, and they could produce wax patterns for investment castings (Wohlers & Gornet, 2014). In the year 2013, the technology of 4D printing first came into being (Miao et al., 2017), and since its inception, this technology has rapidly grown and evolved towards betterment. 4D printing can be considered as one of the disruptive technologies of recent times, capable of producing intricate, stimuli-responsive 3D structures (Fig. 7.1). The main factor that distinguishes 4D printing from 3D printing is the additional dimension of time. Hence, 4D printing is only possible with materials that are temperature/humidity/pressure-responsive along the passage of time. Tissue and organ engineering has been greatly benefitted by the introduction of AM and 4D printing technologies. Sometimes 4D printing has been described as an

FIGURE 7.1 Building blocks of 4D printing development. Adapted from Sun, Y.-C., et al. (2019). 4D-printed hybrids with localized shape memory behaviour: Implementation in a functionally graded structure. Scientific Reports, 1, 113.

7.2 Polymers for 4D printing

FIGURE 7.2 The difference between 3D and 4D printings. Adapted and reconstructed from Bodaghi, M., et al. (2019). 4D printing self-morphing structures. Materials, 12, 81353. Available from

advanced form of 3D printing. The functionality, physical features, including shape, varies with respect to time. Parameters like humidity or temperature may cause changes in the 3D structure with time, and this forms the basis of the additional dimension of 4D printing (Haleem & Javaid, 2019). As with AM, here also CAD involving complex mathematical modeling is used for designing the object to be printed (Momeni, Liu, & Ni, 2017) (Fig. 7.2). The medical world has significantly benefitted from the discovery of 4D printing technology, particularly in the fields of tissue engineering (Hendrikson et al., 2017; Miao et al., 2016; Tamay et al., 2019). Very precise and particular medical devices such as shape-memory based personalized endoluminal devices (Zarek, Mansour, Shapira, & Cohn, 2017) have been successfully manufactured using 4D printing technology. This chapter focuses on summarizing the research reports on 4D printing technology and the polymeric materials used for the application of tissue engineering (Fig. 7.3).

7.2 Polymers for 4D printing AM is a well-known procedure through which layer by layer complex structures can be fabricated. In the case of AM, the 4D printing technology is an up-to-date technology that has drawn potential attention of the modern scientific community



CHAPTER 7 Polymers for additive manufacturing

FIGURE 7.3 Illustrative diagram showing different stimulus for 4D printing and applications of 4D printing in medical filed. Adapted and reconstructed from Morouc¸o, P., et al. (2020). Four-dimensional (bio-) printing: A review on stimuli-responsive mechanisms and their biomedical suitability. Applied Sciences, 24, 9143.

for its high capacity towards the fabrication of complex structures, environmental stimuli-sensitive 3D structures and hence can be significantly used for the application of tissue and organ engineering applications (Kianian, 2017; Miao et al., 2017; Mitchell, Lafont, Hoły´nska, & Semprimoschnig, 2018). In the modern era, there are various smart materials like polymers and polymeric composites, which have been introduced for 4D printing. Among them, some materials to be highlighted are hydrogels, shape memory polymers, elastomer actuators and stimuli-responsive polymers (Roy et al., 2020).

7.2.1 Hydrogels Hydrogels were found to be widely used in 4D printing technology. Hydrogels are cross-linked polymeric networks that have the ability to absorb a large amount of water molecules in their network and also exhibit excellent cytocompatibility (Roy, Maity, Bose, Dhara, & Pal, 2019). The added dimension in the case of 4D printing of stimuli sensitive hydrogels is well defined as the variation of the swelling ratio, which exerts change in the shapes of the materials in response to the external stimuli (Roy, Maity, Dhara, & Pal, 2018; Shiblee, Ahmed, Khosla, Kawakami, & Furukawa, 2018; Zhao et al., 2018). In this regard, temperature and hydration sensitive cellulose-based hydrogels were synthesized and reported,

7.2 Polymers for 4D printing

which has been used as composite ink for 4D printing. For the preparation of cellulose composite hydrogel material, a hydrophilic mixture of carboxy-methyl cellulose polymer, clay platelets, and cellulose fibers were utilized. Further, the hydrophilic mixture was crosslinked (self-crosslinking as well as crosslinking with poly acrylic acid and citric acid) to generate a tissue-engineered scaffold (Mulakkal, Trask, Ting, & Seddon, 2018). Furthermore, a pH-sensitive antimicrobial hydrogel scaffold was also reported, which have been used with stereolithography to produce scaffolds for tissue regeneration. The scaffold exhibited outstanding antibacterial properties against S. aureus. This pH sensitive hydrogel was fabricated using Irgacure 819 (IRG 819) as a photoinitiator and acrylic acid (AA) was crosslinked with copolymer of polyethylene glycol dimethacrylates (PEGMA) using various types of dimethacrylate crosslinkers (Garcia et al., 2018). In this context, there was another report of fabrication of thermoplastic polyurethane hydrogel using 4D printing to produce complex tessellated origami structures. This multimaterial trilayer contained hydrophobic polyurethane materials, which was sandwiched around a hydrophilic polyurethane core. The hydrogel was fabricated using dehydrated techophilic thermoplastic polyurethane (TPU) pellets using a filament extruder with specifications of 2.85 mm ( 6 0.05 mm) diameter at 190 C. This material had unique property to bend and open up depending upon hydration of the scaffold (Baker et al., 2019). In another study, there was a report on ultrafast 4D printing of ionic strength responsive hydrogel. This unique hydrogel was fabricated using visible lightmediated polymerization of monomers (hydroxyethyl acrylate, hydroxyethyl methacrylate, potassium 3-sulfopropylmethacrylate) using IRG 819 as photoinitiator and polycaprolactone diacrylate (PCLDA) as crosslinker. This work demonstrated that the photoinitiator initially created a digital stress circulation in the synthesized 2D polymeric film, postfabrication upon release of that stress, the polymeric scaffold was converted into a 3D scaffold. These hydrogels can potentially be utilized for tissue engineering applications under suitable circumstances (Han et al., 2018; Huang et al., 2017; Roy & Maity, 2021). Another interesting report was obtained for the hydrogel nanocomposites for 4D printing which was the development of a magnetic field responsive hydrogel. This magnetic nanocomposite hydrogel was used as ink with the help of extrusion. The hydrogel was fabricated via polymerization of acrylamide monomer and carbomer (used as a rheological modifier) and Fe3O4 nanoparticles (used as a ferromagnetic substance). This carbomer centric 3D printing method enlightens new directions for advanced level bioprinting (Chen et al., 2019).

7.2.2 Shape memory polymers SMPs are special kinds of polymers that can transform their shape under the influence of some external stimuli (Salimon, Senatov, Kalyaev, & Korsunsky, 2020). In this context, a modern approach microstereolithography was introduced in the field of 4D printing. This methodology helped to generate high-resolution



CHAPTER 7 Polymers for additive manufacturing

shape memory polymers of various materials and architectures. Thus generated highly potential materials were useful for the AM. As an example, a polymer was fabricated and reported using free radical photo polymerization of benzyl methacrylate and crosslinking using various crosslinkers like di(ethylene glycol) dimethacrylate, bisphenol A ethoxylate dimethacrylate and poly (ethylene glycol) dimethacrylate, etc. to form network like structures (Salimon et al., 2020). On the other hand, there was also an example of SMPs that was a photo sensitive polymeric scaffold. This matrix was produced using tert-butyl acrylate and crosslinked with the help of di(ethylene glycol) diacrylate crosslinker using photoinitiator phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO) (Choong, Maleksaeedi, Eng, Wei, & Su, 2017). The synthesized material deformed when cooled and again regained its shape when heated; hence worked very efficiently as shape memory polymer. Another similar kind of work was reported recently that was postsynthesis of the polymeric material; some nanofiller like nanosilica have been introduced to the matrix to improve the overall strength of the material. On the other hand, silica also helps to generate high speed in printing (Choong et al., 2020). Recently, a novel material that helps in the digital light synthesis of shape memory polymers was reported. Interestingly it was fabricated within 30 s, which has significant control in geometries as well as in the shape memory properties. This scaffold had been fabricated using isobornyl acrylate as the monomers and 1, 6-hexanediol diacrylate as the crosslinker. Prior to the fabrication, homogenous mixture of printing precursors has been achieved to confirm the dissociation of the photoinitiator (bis (2, 4, 6-trimethylbenzoyl)- phenylphosphineoxide). For the fabrication of the material, the light of 400730 nm wavelength had been irradiated. Furthermore, the 4D printing was carried out using nano-photonics (Zhang et al., 2019). Likewise, an efficient material containing dynamic imine bonds via crosslinking of methacrylate monomer with 2-(methacryloyloxy ethyl 4-formylbenzoate) utilizing hyperbranched cross-linker was also fabricated and reported. The biggest advantage of this material was its self-healing nature. This was also very efficiently used for 4D printing (Miao et al., 2019).

7.2.3 Elastomer actuators An elastomer is a polymeric material that possesses viscoelastic properties. 4D printed elastomer actuators can efficiently be used in various AM applications like in artificial muscles, bioinspired robots, and so on (Lau, Shiau, & Chua, 2020). In the modern research of elastomer actuators, one unique discovery is liquid crystal elastomer (LCE) ink. The LCE was a new 4D printing programming technique. The LCE ink was developed by mixing 1, 4-bis-[4-(6-acryloyloxyhexyloxy)-benzoyloxy]-2-methylbenzene, n-butylamine and Irgacure 651 at 110 C. The parameter programmed 4D printing opted could be used in wider applications like the development of various software tools (Ceamanos et al., 2020). With the advancement of research, heat-responsive composite hybrid polymeric actuators were fabricated using epoxy-acrylate and also used in 4D printing technology

7.2 Polymers for 4D printing

(Yu et al., 2017). Besides, scaffolds with multifaceted applicability like soft robotics, actuators, shape-changing patterns, chronologically folding box, the hinge were developed using LCE ink was prepared by using aza-Michael addition reaction of acrylate with thiol (Roach, Kuang, Yuan, Chen, & Qi, 2018). In another study, there is an example of the development of a controlled orientation gradient 4D scaffold using 4,40 -Bis(6-hydroxyhexyloxy)- biphenyl and 4-(6hydroxyhexyloxy)cinnamic acid as monomers. These materials were polymerized under N2 atmosphere to form the printable polymeric ink for 4D printing. The scaffold has been used as temperature-responsive 4D printed multiple actuators (Zhang et al., 2019). In this context, a photo-responsive liquid crystalline elastomer actuator containing azobenzene was fabricated using n-butylamine, 1,4-Bis-[4-(6-acryloyloxyhexyloxy)benzoyloxy]-2-methylbenzene, and 4,40 -Bis[9 (acryloyloxy)nonyloxy]azobenzene in 1: 1: 0.12 mole ratio. These 4D printed materials have lifted the objects which possessed many times more weight than their own weight. This behavior demonstrated a high capability to yield effective work. One more advantage of this material was that it could be excited using UV as well as blue lights. The use of blue and UV irradiation permits the fine-tuning of produced forces that could be sustained even in a gloomy situation. Hence, this material has a very well prospect to be used for light-induced robotics applications (Ceamanos et al., 2020).

7.2.4 Thermoresponsive polymers Thermoresponsive polymers alter their behavior in response to alterations in the temperature of the environment (Tamay et al., 2019). At present, stimuliresponsive polymers, especially thermoresponsive polymers, have drawn much attention in the field of 4D printing. In this regard, a recent report was obtained on segmented 3D printed thermogel scaffold for implants, soft robotics, and other biomedical applications. The scaffolds were fabricated using thermopolymer (poly N-isopropyl acrylamide) (NIPAM) and acrylamide (Liu et al., 2019). A 3D printed thermo as well as pH-sensitive scaffolds were developed using triblock polymer of methacrylate poly(ethylene oxide) block poly(propylene oxide), block poly(ethylene oxide) and SL combination (Dutta & Cohn, 2017). Triblock polymer is a special type polymer that is made up of the linear attachment of three blocks of homopolymers. This 3D printed triblock copolymer exhibited a potential dual (pH and temperature) sensitive phenomenon and could be very beneficial in the production of medical devices, as they demonstrate the superior capability to alter its space depending on the change in pH and temperature. Like the triblock copolymers, the interpenetrating polymeric networks (IPN) were also developed and reported. These types of polymeric substances have drawn attention due to their easy synthetic steps and biocompatibility. As an example, alginate and poly NIPAM based IPN was fabricated for 4D printing to fabricate temperaturesensitive smart valves for water regulating purposes (Bakarich, Gorkin, Panhuis, & Spinks, 2015). Another ink for 4D printing was prepared using polyether/



CHAPTER 7 Polymers for additive manufacturing

polyurethane (PE-PU) long-chain polymers, crosslinked with the help of UV exposure to prepare scaffolds. The UV polymerization was achieved with the help of α-ketoglutaric acid as initiator, N, N0 -methylenebisacrylamide as crosslinker and NIPAM as a monomer. Temperature-sensitive hydrogel hinges were also fabricated from several ink constructs. Thus using this simple structure, hydration and heat-responsive, more complex structures and devices can be fabricated in future (Naficy, Gately, Gorkin, Xin, & Spinks, 2017).

7.3 Application of 4D printing technology 7.3.1 Engineered tissue constructs In the recent era, 3D printing has evolved into an incomparable technology for bio-based manufacturing (Ngo, Kashani, Imbalzano, Nguyen, & Hui, 2018; Yan et al., 2018). A number of scientific evidence are present to establish the fact that 3D printing technology has a great potential for engineering artificial organs or functional tissues to heal nonfunctioning, abnormal or necrotic tissues. 4D printing, on the other hand, integrates time-dependent dynamic characteristics to the advanced fabricated platforms for tissue engineering applications (Chu et al., 2020; Miao et al., 2017). Soft tissue regenerative implants Soft tissue anatomically includes skin, adipose, musculoskeletal, liver, lung, kidney, and ocular tissues. Soft tissues are highly flexible, and their functions depend on the mechanical properties of the tissue. All these mechanical properties are again dependent on the composition and arrangement of the extracellular matrix (ECM). Soft tissue damage is a major concern as it may lead to scarring or disfigurement and loss of bodily function. In order to overcome the limitations of current therapies related to damage or diseased soft tissues and organs, various new strategies have been developed for soft tissue engineering (Gokhare et al., 2017; Junk & Kuen, 2016). In a recent study, 4D printed hydrogel was reported for the construction of selffolding and hollow constructs with a very small diameter of 20 μm. Biopolymers like Alginate and hyaluronic acid has been employed here for the hydrogel construct. The constructs were biocompatible and could undergo reversible changes with the change in Ca21 ion concentration. Moreover, the constructs supported the survival of cells with a negligible reduction in cell viability (Kirillova, Maxson, Stoychev, Gomillion, & Ionov, 2017). Another study pointed out the use of 4D printed photocurable silk fibroin (Sil-MA) hydrogel to be used in soft tissue engineering of the trachea. The construct was found to be highly cell-friendly and biocompatible and was printed using digital light processing (DLP). The interior and exterior properties were

7.3 Application of 4D printing technology

modulated in physiological conditions in order to control the shape change of the bilayer 3D printed bilayered Sil-MA hydrogels. Finite element analysis (FEA) simulations were employed in order to explore possible changes within the complex structure. The 4D construct showed proper integration with the host trachea after 8 weeks, and formation of both cartilage and epithelium was noticed (Martinez et al., 2018). One of the recent studies pointed out near-infrared light (NIR) responsive 4D printed nanocomposite for probable use in soft tissue engineering and paving the way towards the 4D printed brain model. A smart epoxy was used with significant shape memory property, which was doped with graphene to generate a nanocomposite that exhibited an exceptional photothermal effect. The synthesized nanocomposite was highly responsive towards NIR stimulus, and its transformation was controllable dynamically and remotely in a spatiotemporal manner. Owing to the high electroconductive and optoelectronic properties of the nanocomposite, the constructed 4D neural cell-laden exhibited exceptional neural stem cell growth and differentiation (Wohlers & Gornet, 2014). Similarly, a NIR light-responsive 4D construct was fabricated using polyethylene glycol diacrylate (PEGDA) by DLP printing process for cardiac tissue engineering. 4D cardiac constructs were fabricated with aligned myofibers and adjustable curvature such that biomimetic structures of the myocardial tissue in both macro- and micro-scale can be obtained. This 4D construct was able to promote uniform cell growth and distribution throughout the curved structure. The 4D construct possessed remotely and active controllable transformation in a spatiotemporal manner and thus making it suitable as a cardiac construct. The 4D printed construct was found to be highly biocompatible when tested using humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), human bone marrow mesenchymal stem cells (hMSCs), and human umbilical vein endothelial cells (hECs) and the cell got uniformly distributed throughout the curvature and exhibited excellent myocardial maturation (Miao et al., 2017). Other than this, 4D printed multiresponsive structures has also been constructed for advances in nerve tissue engineering. Shida Miao; constructed a 4D bio-printed structure using stereolithography (SL)-based technique. Materials used was mostly natural and photo-cross-linkable, like soybean oil epoxidized acrylate, SOEA. In order to achieve multiresponsive 4D construct a combined design of shape memory effect and stress-induced shape alteration was proposed and employed. The shape memory construct derived from natural biopolymers was able to initiate an additional “thermomechanical programming” shape transformation as well, thus pointing towards its multifunctionality. The introduction of nanomaterials amplified the overall 4D effect. A concept of a reprogrammable nerve guidance channel was validated using hMSCs, which in turn can differentiate into neural cell types. Thus this 4D construct with multifunctional characteristics will pave a way towards nerve regeneration (Haleem & Javaid, 2019).



CHAPTER 7 Polymers for additive manufacturing Hard tissue regenerative implants Although bone tissue has the property to heal on its own; however, its selfhealing property depends on the defect size (Szpalski, Wetterau, Barr, & Warren, 2012). Due to this, the healing of large bone defects is still afflicting surgeons (Marelli et al., 2011). In this regards, bone and tissue engineering has been explored vastly (Ansari, 2019). 4D printing technology has been utilized by researchers to fabricate scaffolds for promoting bone and dental healing by creating specific dynamic microenvironments (Khorsandi et al., 2020). Hydroxyapatite (HAp) and polylactide based scaffold was reported to show a temporary shape under compression, and upon temperature change to . Tg, the scaffold could restore its structure. These scaffolds could be used as a selffitting bone implant and could improve medical services (Senatov et al., 2016). 4D printing, on the other hand, gives an option for customized bone tissue engineering scaffolds which can overcome some common problems like implantation of scaffolds in defects with irregular shapes. A 4D printed scaffold with photothermal-responsive shape memory property was constructed using β-tricalcium phosphate/poly(lactic acid-co-trimethylene carbonate) (TCP/ P(DLLA-TMC)) along with incorporating of black phosphorus nanosheets and osteogenic peptide, thus resulting in a nanocomposite scaffold. The scaffold was highly dynamic and was appropriate to serve the purpose of a multifunctional bone tissue engineering scaffold. Application of NR irradiation caused a change in temperature of the scaffold and thus allowing shape reconfiguration and making the scaffold fit irregular bone defects. The inclusion of peptides into the scaffold resulted in enhanced osteogenesis in bone defect sites. A study of the scaffold in an animal model (rat cranial bone defects) showed improved and compact integration of the reconfigurable scaffolds along with enhanced new bone development (Momeni et al., 2017). A 4D printed, biomimetic hierarchical scaffolds showed high biocompatibility and dynamicity, which paved a path for regenerative medicine and tissue engineering. The dynamic scaffold was fabricated using castor oil and polycaprolactone (PCL) triol and was crosslinked using poly(hexamethylene diisocyanate) (PH). The printed scaffold was highly porous, indicating proper cellular growth and differentiation. The scaffolds were found to be highly biocompatible with mesenchymal stem cells (MSCs), and they also exhibited proper attachment, proliferation, and differentiation. High mechanical stability and superior biocompatibility make these types of scaffolds extensively suitable for bone tissue engineering (Miao et al., 2016). Notably, it is important to mention that the addition of functionalized mineral additives (tricalcium phosphate, HAp), decellularized bone matrix and trace elements (Mg, Zn, Sr, Ag, Si, and Sr) into 4D printed scaffolds could further improve their osteoinductive and osteoconductive properties (Kulanthaivel, Agarwal, Rathnam, Pal, & Banerjee, 2021; Qu, Fu, Han, & Sun, 2019; Ribas et al., 2019; Sawkins et al., 2013) (Fig. 7.4).

7.3 Application of 4D printing technology

FIGURE 7.4 Applications of 4D printing in hard tissue engineering. (A) Application of thermosensitive hydrogels for 4D bone tissue regeneration, (B) Application of shape memory and shape responsive material for 4D bone tissue regeneration, (C) Biomimicry for 4D tissue engineering. Adapted from Wan, Z., et al. (2020). Four-dimensional bioprinting: Current developments and applications in bone tissue engineering. Acta Biomaterialia, 101, 2642.

7.3.2 Medical devices Vascular stenosis is a major complication for cardiac patients, and the trending increasing cases of the prevalence has been reported to be a serious threat for the people (Im, Jung, & Kim, 2017). In vascular stenosis, the lumen of the artery has been reported to narrow due to plaque formation along the arterial inner wall, resulting in atherosclerosis (Sigwart, Puel, Mirkovitch, Joffre, & Kappenberger, 1987). The presently available nondegradable metal stents generally used to treat vascular stenosis have been reported to increase the occurrence of restenosis due to the proliferation of intimal smooth cells. To overcome this complication biodegradable vascular stents fabricated using 4D printing technology have been proposed to be an



CHAPTER 7 Polymers for additive manufacturing

excellent solution (Fu, Liu, & Hu, 2018; Huang, Zhang, Scarpa, Liu, & Leng, 2018; Park et al., 2015; Yakacki et al., 2007). In a study, personalized 4D printed stents were developed using shape memory PLA with negative Poisson’s ratio for application as a vascular implant. Fluid-structure interaction and stress distribution during the shape recovery process for the stents were mostly based on the finite element method. This study has also reported that the shape memory behaviors of the 4D vascular stents fabricated were vital in stimulating narrow blood vessels. It was also reported that these 4D stents significantly assisted in the recovery of vascular stenosis conditions (Lin, Zhang, Liu, Liu, & Leng, 2020). A study in the recent past showed the use of both shape memory and shape healing properties of polymer using 3D printing. The ink was prepared using a photocurable resin composed of aliphatic urethane diacrylate (AUD; containing 33 wt.% of isobornyl acrylate) and n-butyl acrylate (BA) and a semicrystalline polymer, that is, PCL. The printed construct was a highly stretchable (600% with in-plane isotropic properties) semiinterpenetrating polymer network (semi-IPN) elastomer and was printed using a direct-ink-write (DIW) approach. The highly stretchable elastomer with the dual property was further demonstrated to be used as a vascular repair device. The unique property of this material makes this a good choice for various biomedical applications as well (Tamay et al., 2019). Similar multifunctional material was developed by Hongqiu Wei A 4D active shape-changing structure was fabricated by the direct wire printing technique. The material used was poly-lactic acid-based ink which was crosslinked using ultra violet rays. Fe3O4 and benzophenone (BP) were further introduced in order to achieve remotely actuated characteristics and excellent shape memory, respectively. The printed material showed excellent shape memory behavior with various configuration transformations, and the addition of iron oxide enhances the property of the material by integration of fast, remotely actuated, and magnetically guidable properties. The multifunctional property and the flexibility of this material make this a perfect candidate for a self-expandable intravascular stent. These types of multifunctional materials pave a new direction for additional development in the field of 4D printing, biomedical devices, soft robotics, microsystems, and beyond (Hendrikson et al., 2017). Other than vascular stents, recent advancements in the fabrication of tracheal stents using thermosetting materials have also been reported. A group demonstrated a heat-driven lumen device as a tracheal stent which becomes functional with the increase in temperature. UV-LED stereolithography printing technique has been used for printing methacrylated PCL precursor based material which turned out to be highly biocompatible and exhibited excellent dynamic property and shape memory (Zarek et al., 2017).

7.3.3 Drug delivery implants Drug release at a suitable time and at the appropriate location is of utmost importance in the area of pharmaceuticals and drug delivery. This is the major emphasis of the drug development industry. 4D printing technologies make it possible to

7.3 Application of 4D printing technology

optimize conditions that can help to control the spatial and temporal delivery of therapeutic agents (Hsu & Jiang, 2019). Utilizing the 4D bioprinting design of various implants can be made possible, which can self-fold or unfold either to engulf and discharge drugs or cells into the system in a programmable way (Dai et al., 2019; Larush et al., 2017). A recent study has reported that NIR-responsive double network shape memory hydrogel could be used to fabricate antimicrobial scaffold. The scaffold has been constructed using Pluronic F127 diacrylate macromer (F127DA), poly(lactide-co-glycolide) (PLGA) and graphene oxide, which served as an energy converter to ultimately convert NIR radiation to thermal energy. The scaffold on testing has been reported to show no cytotoxicity. Restoration of the folded hydrogel into the original shape has been achieved by irradiating it under NIR for about 240 s. The change in the surface area played the controlling factor for drug release property owing to the change in the shape of the scaffold. Thus on distorting the temporary shape, the surface area becomes smaller hence reducing the drug release (Dai et al., 2019). An activated drug delivery system was constructed using alginate fibers and pH-responsive material using 4D printing technologies. The printed material acted as a porous sensor. Simultaneously, alginate fibers loaded with gentamicin has been printed, which acted as drug-eluting stents. Using the printed sensors and the drugeluting stents, a dressing material was fabricated. This dressing material showed potential effects in controlling chronic and acute injuries occurred due to trauma, surgery, or diabetes. The whole system was designed in such a way that upon the exposure of the fabricated material on the wounded or infected site, there will be a change in pH which will be captured by the sensor that will activate the drugeluting stents to release the active pharmacological ingredient at the respective site of the pH change to provide antibacterial activity. Besides, various attempts are going to modify the system to make it more beneficial and to detect more specific bacterial markers (Mirani et al., 2017). A pH-responsive drug release system has also been formulated using DLP technology by using pH-independent fluorescent dye sulforhodamine B as a model drug. The fabricated scaffold has been reported to exhibit improved swelling and rapid drug release under suitable conditions (Larush et al., 2017). Another study exhibited a drug delivery model using shape memory hydrogels with internal structure (SMHs), which was fabricated using pluronic F127 diacrylate macromer (F127DA) and sodium alginate. The 3D printed structure was comprised of a dual network structure: Stable network and reversible network. These shape memory hydrogels were found to be highly biocompatible with fibroblast cells, and the drug-releasing rate was found to be more rapid than other conventional drug-loaded hydrogels (Zarek et al., 2017). All these studies indicate that 4D additive technology provides a way to construct both simple and complex structures that can control localization, release rate, and smart delivery of drugs (Wang & Kohane, 2017). A list of important tissue engineering work related to 4D printing has been summarized in Table 7.1.


Table 7.1 Application of 4D printing in tissue engineering and various medical field. Medical application 4D additive printing in smart stent


Material used



• 4D printing technology can be used to create stents that would stretch and assume the desired form with the aid of the patient’s body heat • This new approach works efficiently to save a patient’s life from a potentially life-threatening operation • Shape changing polymer with respect to time and temperature

Formulation of polycaprolactone (PCL)-based resin for personalized endoluminal medical devices- PCL diol, stannous octoate, isocyanatoethyl methacrylate, 2,4,6-trimethylbenzoyldiphenylphosphineoxide as the photoinitiator Triple shape memory polymers— polyurethane-based filaments, fabricated using fused deposition modeling printing technology

• Personalized endo-luminal medical devices x Since the stent is almost identical to the arcade configuration and placement of the cartilaginous rings, this design strategy based on customized assemblies can reduce migrations, which are a common cause of tracheal stent failure x The shrunk SMP structure’s low profile allows for a less harmful launch • Triple shape memory polymers: x Superior adaptive systems with the ability to self-bend x An elasto-plastic reaction that acts hyper-elastically at low temperatures when acting elasto-plastically at high temperatures in the massive deformation regime x A new method for making self-shrinking/tightening staples. And self-bending grippers/stents

(Bodaghi, Damanpack, & Liao, 2018; Zarek, Mansour, Shapira, & Cohn, 2017)

4D printing technology of organ fabrication

• This new technique will be used to fabricate complex 3D organs • It can be used to print organs using the patient’s own cells and can potentially save a life • It is a potential option for meeting shortages of organs during the crisis

Wood filament combined with acrylonitrile butadiene styrene filamentNylon hectorite clay, nanofibrillated cellulose, and N-isopropylacrylamide monomers or N, N-dimethyl acrylamide, soybean oil epoxidized acrylate,

Multi-material 4D printing

• A layer-by-layer process, by use of UV curable polymer • It’s a brand-new method for printing surgical devices for personalized smart multimaterial printing • The 3D printed body, clearly reveal many body pieces

For supporting dyspnea (a major breathing problem)

• 4D printing technology is now being used as life support to save lives of newborns who are suffering from severe breathing problem (dyspnea)

For linear chain builder (LCB) methacrylate-based polymer (benzyl methacrylate) has been used, LCB and multifunctional oligomers, di-methacrylate, bisphenol, poly (ethylene glycol) for crosslinking purpose ethoxylate di-methacrylate, and Di (ethylene glycol) di-methacrylate has been used PCL

• The fabricated scaffolds will completely restore their original form from a temporary shape set at other temperatures, at human body temperature (37 C) • A high degree of cellular adhesion • A surgical system that is only minimally invasive • Development of 4D printed organs that conform to changing or increasing tissues, particularly for pediatric applications • Tailorable flexibility • Enhanced thermomechanical properties • Multifunctionality

(Miao, Castro, & Nowicki, 2017; Saunders, 2017)

• The 4D printed structures are hollow and porous, designed to spread open as a child grows with age

(Choi, 2015; Haq, 2015)

(Akbari, Sakhaei, & Kowsari, 2018; Ge, Sakhaei, & Lee, 2016)


Table 7.1 Application of 4D printing in tissue engineering and various medical field. Continued Medical application


Material used

• 4D printing technologies have made it possible to fabricate dynamic medical implants that can change shape over time and as babies grow up they don’t face any major problem and can keep breathing

4D printing technology-based tissue engineering and fabrication of smart medical device

• 4D printing gives a potential opportunity to fabricate shapeshifting materials and objects that could be used for human application • It shows a promising prospect when it comes to medical implants and tissue technology, which could modify their form in the body • It’s used to regenerate tissues where the mechanical characteristics of the body alter dynamically when the muscle, bone, and cardiovascular tissue become active

• Polyether urethane • Poly(ethylene glycol) diacrylate, luminol (3aminophthalhydrazide), collagen from calf skin type I

Advantages • The devices improved breathing and expanded to allow the airways • The air splint can degrade within the body after there is suitable growth of the trachea. Hence this will give a new path for curing tracheobronchomalacia • The changes in the morphology of adherent cells could be initiated using a single mechanical stimulus • These 4D printed scaffolds could find use in supporting the regeneration of tissues with dynamically varying mechanical characteristics, for example, cardiovascular tissue • Customized design • These 4D printed scaffolds could be implanted in • Patients by minimally invasive surgery • This technique enables the printing of both cells and programmed calcification together to enable the in vitro reconstruction of cellularized bone defects • Enzyme incorporation


(Hendrikson, Rouwkema, & Clementi, 2017; Mandon, Blum, & Marquette, 2017)

Printing of essential organs like heart, kidney, and liver

• 4D printing in the future will allow the use of intelligent material to manufacture the heart, kidney, and liver. Hence dynamicity will be maintained • Ability to print such components that are very flexible, fit, and match genetically perfect

• 4D printed heart: HeartPrint flex material • 3D printed organs: natural materials like alginate, gullan gum, cellulose, and synthetic materials include PCL, Silicone Pluronic F127 Intensive research is going on 4D printed organs

Advanced skin grafts or artificial skin

• 4D printing has made it possible to print skin graft which looks original and realistic with matching the color complexions of the patients • Also beneficial for patients with severe burn as these printed skin grafts can quickly integrate within the body and start growing like an original

Skin bioprinting: Collagen type I embedded with Mouse NIH3T3 Swiss albino fibroblast and human immortalized HaCaT Keratinocyte cell lines Fibrinogen/collagen hydrogel

• Better visual input and tactile information to comprehend complicated heart abnormalities • Translucent material for the printing enables the inner structures to be easily visualized • The heterogeneous structure may be manufactured at the same time and diverse cell maturations are accomplished • Accurate stratification • Better properties of skin properties • The potential of reconstructing vascular network • Direct cell incorporation in ECM matrix

Fabrication of smart medical devices using 4D printing technologies

• This technique is capable of producing complicated intelligent, 3D-printed medical devices with outstanding functionality • Adjusted according to the time required for the surgical procedure

Semi-IPN based materials: PCL, EBECRYL 8413 SMPs, shape memory ceramics, shape memory gels, and other shape memory hybrids materials, for examples polylactide, thermoplastic polyurethane, and UV-cured thermoset polymers such as VeroWhite Plus RG835 Photocrosslinkable polyethylene glycol

• Remarkable functional characteristics, such as highstrain shape memory and shape memory assisted selfhealing • Extensive material having inplane isotropic characteristics and the objects can be stretched by upto 600% • Heterogeneous 4D printed parts can be printed together

(Gosnell, Pietila, & Samuel, 2016; Yi, Lee, & Cho, 2017)

(He, Zhao, & Zhang, 2018; Khoo, Teoh, & Liu, 2015)

(Castro, Meinert, Levett, & Hutmacher, 2017; Kuang, Chen, & Dunn, 2018; Pei & Loh, 2018; Zhao, Yu, & Li, 2018)


Table 7.1 Application of 4D printing in tissue engineering and various medical field. Continued Medical application

4D printing for complex surgery


• 4D printing technologies are used to generate a haptic model that represents the body’s movement and appearance • For extremely difficult surgeries that other manufacturing technologies cannot accomplish, it may likely be embraced in the future using 4D printing • Producing a model by using various smart material could be made possible with the help of real-time CT and MRI scan which in turn can precisely reproduce any type of body movement • Any type of anatomical details can be depicted in a precise and accurate manner using this technology

Material used

Photopolymer or epoxy resinPhenylbis (2, 4, 6-trimethyl benzoyl) phosphine oxide (as a photoinitiator), Polymer base: -methacrylate-based polymer, Sudan I and Rhodamine B working as photo absorber

Advantages • The joint folding direction can be controlled by the radial orientation of the 4D printed sheets concerning the adjacent structures • In order to enhance clinical outcomes and for more successful procedures, superior spatiotemporal anatomical information could be extracted using this enhanced technology. Thus this would assist surgeons to a large extent • This technique enables the design of time-dependent sequential shape recovery of a structure fabricated using 4D techniques • This technique could be of potential use for denture positioning and denture retention and upgrading the existing dental implants • For a difficult procedure, the surgeon can foresee the problem and better comprehend it


(Chae, Hunter-Smith, & De-Silva, 2015; Hegde & Hsiao, 2016; Javaid, Haleem, & Kumar, 2019; Lee, An, & Chua, 2017)

• Additive manufacturing technologies that could be a major support for dentistry are helping in saving time as well as its cost is also lower • With the use of 4D flow MRI, high-resolution imaging of the complete Fontan circulation could be made possible, including anatomy, and blood flow in just a single 1015 min acquisition • It is conceivable that the surgeon can update the detailed patient-specific heart defect model with interactive anatomy-editing instruments, such as SURGEM and GeoMagic studio • The approaches allow for the use of 4D flow in patients with Fontan circulation for the refining of surgical techniques to quantify many variables of clinically important


CHAPTER 7 Polymers for additive manufacturing

7.4 Conclusion In the fields of tissue engineering and biomaterials, three-dimensional printing is quickly becoming a critical tool. By creating patient-specific devices with the required shape and organization, it transformed the biomaterials industry. Biomedical devices have long used stimuli-responsive materials, such as metals and polymers, and the combination of these two properties creates 4D printing, which introduces incredibly useful, viable, dynamic, and responsive systems in tissue engineering applications. 4D printing is a revolutionary technology. Despite decades of research, only a few responsive materials have been developed for 4D printing and are now being used in tissue engineering. Biocompatible, noncytotoxic, and preferably biodegradable materials are required for this purpose (resorbable). It’s also essential that they’re capable of dynamic processes in physiological environments, as well as displaying certain mechanical strength. The stimulus used for such application must be safe according to the standard and easy to control when applied to the body, which is an important consideration. It’s best to stay away from things like pH extremes and extremely high temperatures. Only a small number of dynamic polymers meet all of the criteria due to the high standards. Furthermore, tissues in nature are exposed to a wide range of stimuli, whereas the majority of materials described to date respond only to a single stimulus. In order to improve 4D printing technology, more effort and expertise should be put into developing new and multifunctional 4D inks. As it stands, 3D and 4D printing methods continue to keep scientists busy as they try to develop new biomaterials and biomedical instruments. While there are many different types of stimuli out there, the materials currently respond to a small subset of them. Thus, the development of new materials with multiple sensitivities for use in enhancing the dynamic nature of devices remains a difficult problem.

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Bioprinting of hydrogels for tissue engineering and drug screening applications


Ece O¨zmen , O¨zu¨m Yıldırım and Ahu Arslan-Yıldız Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey

8.1 Advancements in bioprinting technology Every year, millions of people suffer from tissue or organ deficiency related diseases, and number of donors is far less than the need. Transplantation saves lots of lives; however, immune rejection limits transplantation (Lanza, Langer, Vacanti, & Atala, 2020). Hence tissue-engineering approaches have been developed for treatment of damaged tissues and fabricating artificial organs. Scaffolds with or without cells are fabricated by using conventional or advanced manufacturing techniques, which provide the proper cell culture conditions, and maintain cell viability and proliferation (Langer et al., 1995). In addition to transplantation purposes, tissue engineering approach is used to create 3-dimensional (3D) cell culture systems, which model disease and are utilized for drug discovery. Drug discovery and drug screening studies are generally performed on 2-dimensional (2D) cell culture and animal models. However, in 2D cell culture models, cells grow as a monolayer and do not represent the native 3D tissues efficiently. On the other hand, there are biochemical differences between animal models and human physiology and obtained results cannot be compared with clinical data directly (Onbas, Bilginer, & Yildiz, 2021). To overcome these disadvantages, 3D cell culture model has emerged and is generally used for modeling diseases or drug screening purposes. 3D cell culture models can be fabricated by using both scaffold-free and scaffoldbased techniques. Scaffolds are generally fabricated by freeze-drying (Guzelgulgen, Ozkendir-Inanc, Yildiz, & Arslan-Yildiz, 2021; Rnjak-Kovacina et al., 2015), solvent-casting (Mikos, Bao, et al., 1993; Mikos, Sarakinos, Leite, Vacant, & Langer, 1993), electrospinning (Arica, Guzelgulgen, Yildiz, & Demir, 2021; Bilginer, Ozkendir-Inanc, Yildiz, & Arslan-Yildiz, 2021; Tu¨rker, Yildiz, & Yildiz, 2019), and bioprinting (Arslan-Yildiz et al., 2016; Kang et al., 2016). Compared to other biofabrication methodologies bioprinting is an emerging advanced manufacturing technology, which enables the creation of a designed shape in 3D by printing the bioink material that encapsulates cells or other 

These authors contributed equally and were written in alphabetic order.

Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 8 Bioprinting of hydrogels

biological molecules (du Chatinier, Figler, Agrawal, Liu, & Zhang, 2021; Mei, Rao, Bei, Liu, & Zhao, 2021; Singh, Choudhury, Yu, Mironov, & Naing, 2020). In the field of tissue engineering and regenerative medicine, bioprinting is a widely used advanced manufacturing technique with its reproducibility, high throughput, and controllability in an automated way (Gungor-Ozkerim, Inci, Zhang, Khademhosseini, & Dokmeci, 2018). It is possible to produce tissue scaffolds with bioprinting technology, as well as to produce functional and living 3D structures by encapsulating cell into bioink (Hospodiuk, Dey, Sosnoski, & Ozbolat, 2017; Matai, Kaur, Seyedsalehi, McClinton, & Laurencin, 2020). Creating a scaffold or functional tissue via bioprinting process requires utilizing the (1) bioprinting strategies, (2) bioprinting parameters, and (3) bioinks as illustrated in Fig. 8.1. Based on the working principle of bioprinting strategies, either laser-assisted bioprinting (LAB), droplet bioprinting, or extrusion-based bioprinting, the intended use and bioink requirements are varied (Arslan-Yildiz et al., 2016; Murphy & Atala, 2014; Pati, Gantelius, & Svahn, 2016). In LAB: a laser beam that is pulsating in a controlled time span, a surface where cells and bioinks will be positioned, and an absorption layer are utilized for bioprinting of cells and 3D structures. The absorption layer, which is made of gold or titanium, is transparent to the laser beam and covered with bioink material to encapsulate and protect cells and other biological molecules such as proteins (Pati et al., 2016). Resolution of the LAB can be tuned by changing the energy and the spot diameter of the laser beam. The depth of absorption layer, thickness of bioink layer, and

FIGURE 8.1 A schematic representation of the major components of bioprinting.

8.1 Advancements in bioprinting technology

bioink viscosity are the other significant parameters for the efficiency of LAB operation. By tuning the required parameters, LAB allows reaching microscale resolution with computer aided geometrical control (Guillotin et al., 2010). Since LAB is a nozzle-less type of bioprinting operation, the bioprinting of high cell densities and high viscosity of bioinks without causing any clog formation can be achieved (Arslan-Yildiz et al., 2016; Hribar, Soman, Warner, Chung, & Chen, 2014). On the other hand, it is not possible to use LAB for bioprinting of large 3D structures due to the limited spot area of the laser beam. Presence of metallic residues in postprinting structures, the relatively higher price of device, and higher cytotoxicity because of UV exposure are other drawbacks of LAB (Pati et al., 2016). In tissue engineering, LAB have several utilizations for cell printing (Guillotin et al., 2010), patterning (Bourget et al., 2016; Catros et al., 2011; Devillard et al., 2014), modeling (Michael et al., 2013), and regenerative medicine applications (Guillemot et al., 2011; Keriquel et al., 2017; Ke´roure´dan et al., 2019). Droplet bioprinting, in other words inkjet-based bioprinting, is a noncontact technique which is used for printing multiple cells and proteins layer-by-layer by depositing small droplets on the targeted surfaces (Pati et al., 2016). According to the droplet generation strategy, droplet bioprinting is classified into two subgroups: thermal and piezoelectric droplet bioprinting. Droplet bioprinting has been preferred during the last decades due to its high printing speed, high resolution, and cost-effectivity (Gurkan et al., 2014). High cellular viability is achieved by this approach, however it is not suitable for printing high cell densities and highly viscous hydrogels. Since only low viscosity hydrogels can be printed, the created 3D structures cannot provide desired mechanical properties. This limits the efficient achievement of completely functional and 3D usage suitable constructs (Ferris, Gilmore, & Wallace, 2013; Kim, Choi, Kim, Choi, & Cho, 2010). In the field of tissue engineering, droplet bioprinting is mostly utilized for modeling (Christensen et al., 2015; Xu, Chai, Huang, & Markwald, 2012), stem cell research (FaulknerJones et al., 2015; Gurkan et al., 2014), drug screening (Rodrı´guez-De´vora, Zhang, Reyna, Shi, & Xu, 2012), and cancer studies (Fang et al., 2012; Xu et al., 2011). Extrusion-based bioprinting is the third bioprinting approach that aims to print cells encapsulated by hydrogel bioink as designed 3D structures onto target surface. Extrusion-based bioprinting can be classified into two subgroups on the basis of working principle: pneumatic, and mechanical (screw and piston). In each principle, bioink is printed by applying pressure to overcome the surface tension on the nozzle tip. Extrusion-based bioprinting allows printing of high cell density with an adequate cell viability and high viscosity bioinks within a sufficient speed. Properties of 3D construct can easily be tuned by changing pressure, printing speed, layer height, nozzle diameter, etc. Despite these advantages, the printing of high viscosity bioinks requires higher pressure, which results in a decrease in cellular viability, and causes deformation in cellular integrity. In addition, printing resolution is worse than the aforementioned approaches, it is nearly about 200 μm (Malda et al., 2013). Bioprinting speed is higher in this approach



CHAPTER 8 Bioprinting of hydrogels

compared to other methodologies and this technique allows printing constructs in desired dimensions without any limitation. Mechanical properties of printed constructs can be improved by using highly viscous bioinks. In literature, there are studies utilizing the extrusion-based bioprinting with the purpose of cell printing (Almeida et al., 2014; Horva´th et al., 2015), 3D cell culture (Yu, Zheng, Chen, Chen, & Hu, 2014), disease modeling, and cancer research (Faulkner-Jones et al., 2015; Gebeyehu et al., 2021). In addition to the bioprinting strategies, bioprinting parameters are also crucial in performing successful bioprinting of bioink materials (Webb & Doyle, 2017). They are classified into three major groups; (1) process parameters which include pressure, distance, flow rate, laser spot diameter, tip diameter, layer height, etc.; while concentration, viscosity, and crosslinking are included in (2) bioink parameters; and the third group includes (3) environmental parameters, like temperature. Process parameters for bioprinting applications vary for each bioprinting strategy. In LAB, volume of the printed material depends directly on the laser pulse energy although laser wavelength, laser beam focus diameter, focal distance, and velocity of the scanning mirrors are parameters affecting printing. During printing via LAB, the amount of energy required to provide microdroplet ejection to occur is known as the threshold energy and should be in range of 120 μJ/pulse (Guillemot, Souquet, Catros, & Guillotin, 2010). In droplet bioprinting, due to thermal actuation that is achieved by a heating element, and vapor bubble that is obtained from the temperature increase, the temperature is the most important process parameter that affects the resolution. For piezoelectric droplet bioprinting, a voltage is applied to the piezoelectric material, and by this way a droplet is formed. Therefore by changing the applied voltage, the size and shape of the printed droplet can be tuned (Cui, Nowicki, Fisher, & Zhang, 2017). On the other hand, extrusion pressure, nozzle diameter, flow rate, and speed of movement are the most influential process parameters for resolution of extrusion-based bioprinting. The volume of extruded bioink raises with increasing pressure and flow rate. Reduction in nozzle diameter and increase in movement speed results in a decrease in ejected bioink volume and increase in resolution (Ng, Yeong, & Naing, 2016; Webb & Doyle, 2017). Bioink is the third and most important component of bioprinting process. There are two types of bioinks classified; scaffold-free bioink which includes only cell aggregates and biological molecules, and scaffold-based bioink which contains synthetic or natural polymeric material for encapsulation of cells (Hospodiuk et al., 2017).

8.2 Bioinks Bioinks serve as a scaffold and can carry living cells, biochemical factors, extracellular matrix (ECM) components, as well as proteins. Success of the bioprinting

8.2 Bioinks

mostly depends on the properties of bioinks. Bioink properties, bioink concentration, rheological properties of bioink, viscosity and surface tension are studied under solution parameters for bioprinting applications. The viscosity of the bioink can be in a range between 30 mPa/s and 60 3 107 mPa/s (Mandrycky, Wang, Kim, & Kim, 2016) where the viscosity range of the bioink should be 1300 mPa/s for LAB, and 3.512 mPa/s for droplet bioprinting to avoid clog formation through the nozzle and tip. In extrusion-based bioprinting applications, non-Newtonian fluids whose viscosity depends on the shear rate are preferred. Hydrogels that have shear thinning property become extrudable via the alignment of polymer chains by pressure application (Jungst, Smolan, Schacht, Scheibel, & Groll, 2016). These extrudable shear-thinning hydrogel bioinks can preserve their shape and postprinting fidelity. Increase in hydrogel concentration causes an increase in viscosity, which results in an increase in printing consistency and leads to a more controllable printing of fine filaments by increased printing pressure (Paxton et al., 2017). However, an increased concentration and viscosity can cause clogging in the nozzle of the extruder (Kyle, Jessop, Al-Sabah, & Whitaker, 2017). On the other hand, low viscosity and concentration may cause free flowing of bioink from the nozzle without application of pressure and may lead to soft constructs that cannot sustain their shape after printing (Duan, Kapetanovic, Hockaday, & Butcher, 2014). Therefore the viscosity of the bioink should be in the proper range as aforementioned. Bioinks should also meet some other requirements depending on the applied bioprinting strategy. As summarized in Fig. 8.2, bioinks should have shear thinning and thixotropic behavior for extrusion-based bioprinting applications. Low surface tension and low adhesion properties are required for bioink to be bioprintable as a continuous filament. Rapid gelation and shape retention abilities are also needed to retain the designed structure postprinting (Hospodiuk et al., 2017). In droplet bioprinting, low viscosity and nonfibrous nature are important

FIGURE 8.2 Bioink properties and requirements for different bioprinting strategies (Reprinted by copyright permission from, Hospodiuk et al., 2017).



CHAPTER 8 Bioprinting of hydrogels

requirements for the bioink that is going to be used, to eject droplets and prevent continuous filament formation (Gudapati, Dey, & Ozbolat, 2016; Hospodiuk et al., 2017). The bioink also should have rapid gelation and moderate surface tension properties so that the surface tension of bioink could be overcome for easy droplet formation. These properties make a bioink extrudable under low pressure, so that the cellular viability would not be affected by the application of high pressure. On the other hand, fast gelation mechanism to solidify without causing any spreads is the most important property of the bioink that is required for the LAB strategy. Bioink should also have the ability to adhere onto the intermediate layers, and it should possess low surface tension and viscoelasticity (Wang, Jin, Dai, Holzman, & Kim, 2016). Compared to other strategies the main difference in LAB is the bioink that should have the ability to absorb the kinetic energy supplied by laser beam pulsation. In the case of photopolymerization involved LAB applications, bioink should be photocurable. Moreover, it is crucial for the photo-initiator to be nontoxic and water-soluble to conveniently use the bioink for tissue engineering applications. Bioink should also possess the proper stability and the appropriate mechanical strength for the intended use (Hospodiuk et al., 2017). In addition to each aforementioned property, bioinks should be soft and fluid enough to be manipulated easily during printing process, and hard enough to preserve the printing pattern after printing (Leberfinger et al., 2019). In addition to this, bioink must also promote cell adhesion, be nontoxic, biocompatible, insoluble during cell culture. Depending on the application, biodegradability of the bioink might also be an important property in this context. Besides, it is important that the bioink is easily processed, cost-effective, and commercially available (Hospodiuk et al., 2017). In 3D bioprinting applications, hydrogels that are natural or synthetic polymeric structures are one of the most common bioink materials. The choice of hydrogel to be used as a bioink depends on the rheological properties of the hydrogel and the bioprinting strategy (Hospodiuk et al., 2017). Hydrogel bioinks will be discussed in detail in the following section.

8.3 Hydrogel bioinks Hydrogels are 3D, crosslinked, network-like polymeric structures (Ahmed, 2015). Either chemical or physical crosslinking can be achieved by formation of covalent bonds or physical cohesion forces such as hydrogen bonding, van der Waals forces, or hydrophobic interactions. Hydrogels are classified into two categories as chemical and physical hydrogels based on their crosslinking features (Maitra & Shukla, 2014). They can absorb great amounts of water compared to their mass but remain insoluble because of their crosslinked nature (Peppas, Bures, Leobandung, & Ichikawa, 2000). Due to these desirable properties

8.3 Hydrogel bioinks

(e.g., high amount of water holding capability, biocompatibility, tunable biodegradability, porous structure, elasticity, etc.), hydrogels have great potential to be utilized in biological applications, especially in tissue engineering (Hong et al., 2020; Serafin, Murphy, Rubio, & Collins, 2021; Su et al., 2021) and drug screening studies (Gebeyehu et al., 2021; Monteiro, Gaspar, Ferreira, & Mano, 2020; Xie, Gao, Fu, Chen, & He, 2020). Hydrogels can be classified into two main groups as natural and synthetic, based on polymer sources (Ahmed, 2015). Natural hydrogels are derived from natural polymers or decellularized tissues. They have many advantages such as biocompatibility, biodegradability, and low toxicity (Catoira, Fusaro, Di Francesco, Ramella, & Boccafoschi, 2019). Hydrogels derived from natural polymers are commonly preferred in tissue engineering and regenerative medicine applications, since they have similar chemical/biological content and physical structure of ECM as human tissues (Vieira, da Silva Morais, Silva-Correia, Oliveira, & Reis, 2017). Natural polymers can be grouped as protein-based polymers and polysaccharide-based polymers. While, collagen, gelatin, elastin, fibrin, and silk fibroin are common examples of protein-based natural polymers, alginate, glycosaminoglycans (GAGs), chitosan, and cellulose can be given as examples of polysaccharide-based polymers (Catoira et al., 2019). Beside the natural ones, synthetic polymer-based hydrogels are widely studied in the literature, due to their tunable properties (Koksal et al., 2020). Compared to natural hydrogels, the most important difference is that synthetic hydrogels can be engineered to have the desired mechanical features depending on the application (Chai, Jiao, & Yu, 2017). Considering the properties of natural and synthetic hydrogels individually, both have advantages and disadvantages. To overcome the disadvantages, composite hydrogels that are composed of two or more polymers, are frequently utilized. When different polymers are combined as composite hydrogels, the composite hydrogel may exhibit different features and different physicochemical properties than the individual polymers that are blended. Therefore, composite hydrogels are mostly preferred to obtain desired features for some specific applications (Buwalda, 2020; Rodrı´guez-Rodrı´guez, Espinosa-Andrews, VelasquilloMartı´nez, & Garcı´a-Carvajal, 2020). Hydrogels are complex materials, which consist of solid (a crosslinked polymer network) and liquid (water) components. The amount of the solid and the liquid in hydrogel content affects the final features of the hydrogel. Therefore water-holding capacity is one of the most important properties of a hydrogel. Hydrogels are generally defined by their degree of swelling when exposed to water. Hydrogel swelling depends on polymerwater interactions. Fundamentally, more hydrophilic polymers form stronger interactions with water. There are three forces that are important to expand the hydrogels network; polymerwater interactions, electrostatic interactions, and osmotic pressure which are called swelling forces. Basically, swelling of a hydrogel can be defined as limited solubility. Elastic forces, which originate from the network crosslinking, prevent the complete dissolution of the polymer. The balance between elastic forces and



CHAPTER 8 Bioprinting of hydrogels

swelling forces determines the equilibrium which controls the hydrogel’s swelling. Swelling properties of a hydrogel vary based on many factors such as polymer source, network density, nature of the solvent, polymer solvent interactions, etc. The swelling capacity of a hydrogel depends on the amount of space between polymer chains in the hydrogels’ network that are available to accommodate water. The amount of this space can be regulated by crosslink density. Crosslink density is determined by the concentration of the crosslinker that is used in the crosslinking process. Using high concentration of the crosslinker in the crosslinking process would cause shorter distances between two crosslinks on the same polymer chain. Therefore the amount of space for holding water would decrease with the increasing crosslink density (Peppas, 2010). Swelling of hydrogels are generally expressed as swelling ratio by the following equation: Sð%Þ 5 Wt W20W0 3 100, where W0 is the dry weight of the hydrogel (initial weight) and Wt is the weight of swollen gel at given time (t) (Imren, Gu¨mu¨s¸derelio˘glu, & Gu¨ner, 2006). Mechanical properties are also important parameters for the final hydrogel product. Hydrogels should have mechanical features that would let them maintain their physical structure for a certain period of time during a specific application. Hydrogels are highly popular biomaterials in biomedical applications due to their high water holding capacity, softness, and elasticity (Calo´ & Khutoryanskiy, 2015). However, they have weak mechanical properties, especially in their swollen state (Chen et al., 2016). To improve mechanical properties of hydrogels, several methodologies had been used. For example, desired mechanical strength could be achieved by tuning the degree of crosslinking. Increasing the crosslinking density makes the hydrogel stronger. However, if the crosslinking density is too high, it decreases the elongation property of the hydrogels and causes it to have a more brittle structure (Mishra, Rani, Sen, & Dey, 2018). Another strategy to improve mechanical properties of hydrogels is to blend them with other materials such as nanoparticles (Dannert, Stokke, & Dias, 2019) and nanofibers (Hassanzadeh et al., 2016). Zaragoza et al. used silica nanoparticles to improve mechanical properties of polyacrylamide hydrogels. Their composite system showed that interaction between nanoparticles and polymers resembles a pseudo crosslink. This polymer-nanoparticle interaction provided an increase in elastic modulus and enhanced the mechanical properties of the hydrogel (Zaragoza, Fukuoka, Kraus, Thomin, & Asuri, 2018). Jang et al. used nanofibers to improve strength and durability of hydrogel. In that study nanofibers were electrospun into thin hydrogel solution. Results showed that the comprehensive strength and stiffness of nanofiber-reinforced hydrogel were enhanced to B221% and B434% compared to pristine hydrogel (Jang, Lee, Seol, Jeong, & Cho, 2013). On the other hand, interpenetrating polymer networks (IPNs) are commonly used to provide mechanical strength to hydrogel. Basically, IPNs are defined as a composite structure that is formed by two or more polymer networks interlaced with each other (Karak, 2009). Suo et al. designed an IPN by using GelMA and chitosan hydrogels to improve the mechanical properties. Characterization results of this study showed that IPN structure improved the mechanical features of

8.3 Hydrogel bioinks

GelMA-chitosan composite compared to the pristine chitosan or GelMA (Suo et al., 2018). Biocompatibility is another important feature of hydrogels. It is important to maintain biocompatibility while fabricating materials for biomedical applications. One of the most common and clear definitions of biocompatibility is the ability of a material to perform within an appropriate host response in a specific application (Williams, 1987). To investigate the biocompatibility of materials, several tests can be performed including in vitro cell viability assays, animal trials, etc. (de Moraes Porto, 2012). Biocompatibility of hydrogels is extremely important for tissue engineering and regenerative medicine applications due to the interaction of natural tissue and hydrogels. In crosslinking step sometimes harsh and toxic chemicals are used as a crosslinker, which affects biocompatibility of the hydrogel adversely. Moreover, initiators, organic solvents, stabilizers, emulsifiers, and unreacted monomers which are utilized in polymerization and hydrogel synthesis may be toxic to the host. In this case purification step becomes crucial to remove those toxic residuals from hydrogels (El-Sherbiny & Yacoub, 2013). Biodegradation of hydrogel is a key parameter for the intended use. In tissue engineering applications, controlled biodegradation is mostly expected from hydrogel scaffolds. Hydrogel scaffolds should degrade simultaneously with the secretion of native ECM from cells. Scaffolds provide mechanical stability to cells during tissue formation. If the scaffold degrades before the formation of ECM, cells lose their supporting material and cannot form the expected 3D tissue structure properly (Madduma-Bandarage & Madihally, 2021). Biodegradation behavior of hydrogel scaffolds can be tuned by blending two or more biopolymers that have different degradation rates. Moreover, crosslinking density is a significant factor, which influences the biodegradability of hydrogels. High crosslinking density decreases the biodegradation rate of the hydrogels (Caliari & Harley, 2011). Rapid gelation ability of a hydrogel is an important parameter for printability as it was for bioinks and can be achieved by physical or chemical crosslinking. Physical crosslinking ability eases the gel formation by reducing the risk of chemical residues and does not affect the biocompatibility of the hydrogel. Physical crosslinking of hydrogels can be achieved by self-assembly, hydrophobic interactions, and hydrogen bonding. It provides proper environment for gelation and cell encapsulation (Jungst et al., 2016). Ionic crosslinking is the formation of hydrogel network electrostatically in the presence of opposite ionic charges. Ionic crosslinking is a reversible type of crosslinking mechanism. In addition, hydrogen bonding and hydrophobic interactions can be manipulated by changing the temperature of the hydrogel. The viscosity and rheological behavior of gels can be changed by increasing or decreasing temperature by this manner. Some gels start to show organized chain conformation with decreasing temperature and develop physical gel properties (Janmaleki et al., 2020). On the other hand, in some gels that have hydrophilic and hydrophobic parts, increasing temperature favors the gelation, since hydrophilic parts dissolve at lower temperatures the viscosity of the gel decreases. For these gels, an increase in temperature causes the polymeric



CHAPTER 8 Bioprinting of hydrogels

interactions to be stronger than hydrophobic interactions, and this results in the gelation of hydrogel (Rodrı´guez-Herna´ndez, Pe´rez-Martı´nez, Gallegos-Infante, Toro-Vazquez, & Ornelas-Paz, 2021). The other gelation mechanism of hydrogels is chemical crosslinking, which is achieved by covalent bonding, and provides more mechanical strength to the hydrogels. Crosslinkers or photo-initiators are generally used for chemical crosslinking. However, the presence of crosslinkers can affect the biocompatibility of hydrogels, which is a highly undesired property especially for tissue engineering applications (Naahidi et al., 2017). A higher crosslinker and photo-initiator concentration increase the mechanical strength of crosslinked hydrogel and toxicity while decreasing the swelling property. By controlling all parameters, proper crosslinker and photo-initiator concentration can be tuned, and cytotoxicity can be decreased (Fedorovich et al., 2009; Hennink & van Nostrum, 2012). Hydrogels to be used as bioink should have some of the defined features as described above. In bioprinting applications of tissue engineering, as illustrated in Fig. 8.3, natural, synthetic, and hybrid hydrogels have been commonly utilized

FIGURE 8.3 (A) Natural hydrogel bioinks: bioprinted alginate hydrogel bioink as a hollow cylinder (left); bioprinted hyaluronic acid before crosslinking (right); (B) Synthetic hydrogel bioinks: bioprinted pluronic F127 hydrogels; (C) Hybrid hydrogel bioink: Lattice structure of chitosan modified with meth acryloyl groups (CHIMA) and acrylamide (AM). (A, left) Reprinted by copyright permission from Gao, He, et al. (2015); (A, right) Reprinted by copyright permission from Kiyotake et al. (2019); (B) Reprinted from Suntornnond et al. (2016); (C) Reprinted by copyright permission from He et al. (2021).

8.4 Applications of hydrogel bioinks

during last years (Arslan-Yildiz et al., 2016). Gao and coworkers used alginate hydrogels as bioink for bioprinting of tubular structures that are based on the fusion of hollow fibers (Fig. 8.3A, left). According to mechanical tests and cell viability results, the bioprinted structures produced by hollow alginate filaments were promising to overcome drawbacks about nutrient delivery in tissue engineering applications (Gao, He, Fu, Liu, & Ma, 2015; Gao, Schilling, et al., 2015). In a study by Kiyotake et al., hyaluronic acid (HA) hydrogels were used to characterize the printability of bioink, based on different properties including viscosity, yield stress, and storage modulus recovery. Fig. 8.3A (right) illustrates the printed hydrogel structures with different concentrations of HA (8% and 10%, respectively). As shown in Fig. 8.3A, polymer concentration had affected the printability of bioink. While the bioprinted structure that included 8% HA had clean and defined edges, the structure that produced by 10% HA bioink had sharper, fractured, and irregular edges (Kiyotake, Douglas, Thomas, Nimmo, & Detamore, 2019). In addition to natural hydrogels, synthetic hydrogels are commonly used as bioink material in literature (Aydogdu et al., 2019; Kim, Kim, Hong, Park, & Park, 2021; Piluso et al., 2021). Fig. 8.3B shows bioprinted pluronic F127 hydrogel structures. The main goal of this proof-of-concept paper is to investigate the effects of printing parameters on the properties of a simple and accurate model and to contribute to the development of new bioinks (Suntornnond, Tan, An, & Chua, 2016). Besides the usage of natural and synthetic hydrogels individually, hybrid hydrogels are used to improve properties of bioinks, such as shape fidelity and mechanical strength. He and coworkers modified chitosan by using methacryoyl group and combined chitosan with acrylamide hydrogel to improve poor mechanical stability. Mechanical strength and stability could have been tuned by changing the concentration of acrylamide. As depicted in Fig. 8.3C, acrylamide provided elasticity to the bioprinted structures and they were not damaged when they were stretched (He et al., 2021). In another study, Bednarzig and coworkers developed a promising bioink that consisted of alginate di-aldehyde and gelatin for biofabrication of colorectal tumor-like structures. Developed hybrid hydrogel bioink provided mechanical strength and better cell attachment to the scaffolds (Bednarzig et al., 2021). The aforementioned studies and more are summarized in Table 8.1.

8.4 Applications of hydrogel bioinks Bioprinting technology has a wide-range utilization field like tissue engineering and regenerative medicine (Aljohani, Ullah, Zhang, & Yang, 2018; Gao & Cui, 2016), cancer research (Chaji, Al-Saleh, & Gomillion, 2020), high-throughput assays (Mazzocchi, Soker, & Skardal, 2019), transplantation (Ozbolat, 2015; Ravnic et al., 2017), and drug screening (Lee, Abelseth, De La Vega, & Willerth, 2019; Ma et al., 2018) as illustrated in Fig. 8.4. Printing of designed scaffold and



CHAPTER 8 Bioprinting of hydrogels

Table 8.1 Hydrogels used as a bioink for 3D bioprinting. Material

Bioprinting strategy

Gelatin methacryloyl (GelMA) Poly 3,4ethylenedioxythiophene: Polystyrene sulfonate (PEDOT:PSS)

Pneumatic extrusion 3D bioprinting

Tyramine-modified poly (γ-glutamic acid) [hyaluronic acid (HA), alginic acid, cellulose nanofibrils, methylcellulose] Pluronic F127

Extrusion-based 3D printing

Pneumatic extrusion-based bioprinting

Chitosan Acrylamide

Laser assisted bioprinting

Polyethylene glycol (PEG)

Extrusion-based bioprinting

Chitosan Guar gum

Extrusion-based 3D bioprinting

Gly-Arg-Gly-Asp (GRGD) peptide modified Pluronic-F127 hydrogel Phenylboronic acidmodified laminarin Alginate

Self-fabricated extrusion-based 3D bioprinting system Dual-head extrusion 3D bioprinter



• Tunable mechanical property • Tunable conductivity • High resolution • High cytocompatibility • Promising for complex structures • High printability • High cytocompatibility • High mechanical strength

Spencer et al. (2019)

• Model for developing novel bioinks • Proof of concept • High mechanical strength and stability • High cytocompatibility • Smooth and uniform cylindrical strand fabrication • High shape fidelity • High cell viability • Well defined cell morphology after bioprinting • Promising to fabricate complex 3D structures • Good printability • Potential to be used in the field of the health care applications • Stable printability • Solid configuration • High cell viability

Suntornnond et al. (2016)

• Bioprinting under physiologically relevant conditions

Amaral, Gaspar, Lavrador, and Mano (2021)

Kim et al. (2021)

He et al. (2021) Piluso et al. (2021)

Cleymand et al. (2021)

Chen et al. (2021)


8.4 Applications of hydrogel bioinks

Table 8.1 Hydrogels used as a bioink for 3D bioprinting. Continued Material

Bioprinting strategy

Alginate Gelatin

Inkjet bioprinting


Inkjet bioprinting


Extrusion-based bioprinting


Extrusion-based bioprinting Extrusion-based bioprinting

Gellan gum Poly (ethylene glycol) diacrylate (PEGDA)

Alginate di aldehyde Gelatin

Extrusion-based bioprinting

Gelatin Alginate

Extrusion-based bioprinting


Extrusion-based bioprinting

Advantages • Suitable rheological properties for bioprinting • Homogeneous cell distribution (postprinting) • Good printability • High cell viability • Potential to mimic native tissue • Improved mechanical properties • Precise cell deposition • Layer-by-layer approach without extra steps for cell encapsulation • Potential for nutrient delivery • High cell viability • High mechanical strength • Proof of concept • Excellent printability with living cells • Improved mechanical strength • Decreased shear stress (shear thinning property) • High mechanical strength • Improved cell attachment • Controllable cell orientation and differentiation • Rapid stabilization of printed filaments • Bioprinting without supporting material


Jiao, Lian, Zhao, Wang, and Li (2021) Gao, Schilling et al. (2015)

Gao, He et al. (2015)

Kiyotake et al. (2019) Wu et al. (2018)

Bednarzig et al. (2021)

Distler et al. (2020) Ouyang, Highley, Rodell, Sun, and Burdick (2016)



CHAPTER 8 Bioprinting of hydrogels

FIGURE 8.4 Application areas of bioprinting technology. Compiled by copyright permission from A´vila, Schwarz, Rotter, & Gatenholm (2016), Kim et al. (2020), Park et al. (2017), Si et al. (2019), Xie et al. (2021), Axo A2, Axolotl Biosystems Ltd, Turkey.

controlled positioning of the cells overcome most of the disadvantages of other scaffold production methodologies (Ozbolat, Peng, & Ozbolat, 2016). Bioprinting of living tissue is easily and more practically achieved by using hydrogel-based bioinks. The convenience of hydrogels for each bioprinting strategy and encapsulation of cells make them proper bioink material for bioprinting of functional tissues, tumor models, and tissue grafts (Sun et al., 2020). Bioprinting of tissue-like structures have been successfully performed for years. In this section, the applications of bioprinted hydrogels will be discussed for tissue engineering and drug screening purposes. The creation of functional tissues or organs can be achieved by using scaffoldfree or scaffold-based techniques. In bioprinting applications, both techniques can

8.4 Applications of hydrogel bioinks

be performed by using the proper bioink. Cell pellets, aggregates, and tissue strands are used for bioprinting of scaffold-free constructs (Hospodiuk et al., 2017). On the other hand, hydrogel bioinks are utilized to create scaffolds for cell seeding and create living tissue consisting of bioprinted cells and scaffolds by encapsulating cells into bioink. In literature, as given in Fig. 8.5 there are varied applications of 3D bioprinted hydrogel for bone (Abdollahiyan, Oroojalian,

FIGURE 8.5 (A) Bioprinting of alginate bioink for bone tissue engineering application (right), micro-CT image of bioprinted 3D cell-laden mineralized scaffold (left); (B) Bioprinting of nanofibrillated cellulose and alginate for patient-specific auricular cartilage regeneration (left), 28-days culture of human nasal chondrocytes-laden hybrid bioink (right); (C) 3D bioprinted facial skin reconstruction by using polyurethane (PU), keratinocyte and fibroblast-laden hydrogels composed of hyaluronic acid (HA), glycerol, gelatin, and fibrinogen; (D) bioprinted contractile cardiac tissue composed of fibrin-based composite hydrogel bioink; (E) transected rat spinal cord, and bioprinted iPSC-derived spinal NPCsladen scaffold for spinal cord regeneration by using alginate and fibrin-based bioink, and its top and side-view images. (A) Reprinted by copyright permission from Zhang et al. (2020); (B) Reprinted by copyright permission from A´vila et al. (2016); (C) Reprinted by copyright permission from Seol et al. (2018); (D) Reprinted by copyright permission from Wang et al. (2018); (E) Reprinted by copyright permission from Joung et al. (2018).



CHAPTER 8 Bioprinting of hydrogels

Mokhtarzadeh, & de la Guardia, 2020; Salah, Tayebi, Moharamzadeh, & Naini, 2020), cartilage (Daly et al., 2017; Raut, Agrawal, Bagde, Fulzele, & Syed, 2021; You, Eames, & Chen, 2017), cardiac (Liu et al., 2021; Wang et al., 2021), neural (Lee et al., 2018; Yu, Zhang, & Li, 2020), skin (Weng et al., 2021), and vascular tissue engineering (Zhang & Khademhosseini, 2020) and listed in Table 8.2.

8.4.1 Bone tissue engineering 3D bioprinting is an advantageous methodology for production of anatomically designed tissue constructs to replace bone and trigger remineralization. The bioink is expected to possess osteoinductive and osteoconductive properties. In literature, there are varied studies reporting the bioprinting of hydrogels for bone tissue regeneration (Abdollahiyan et al., 2020). Demirtas et al., bioprinted MC3T3-E1 preosteoblast cells by encapsulating them into chitosan and nanostructured hydroxyapatite (HA) composite hydrogel. The rheological behavior of chitosan and HA were suitable for extrusion-based bioprinting and bioink induced mineralization, providing good mechanical property, and increased cellular viability. Storage modulus was 874 kPa and elastic modulus was 14.97 6 3.99 kPa which is in a suitable range for bone tissue engineering (Demirta¸s et al., 2017). In another study, Bendtsen and coworkers also bioprinted a composite hydrogel consisting of alginate, polyvinyl alcohol (PVA) and HA by using extrusion-based bioprinting. Viscoelastic properties of alginate/PVA/HA composite hydrogel resulted in high shape-fidelity while alginate and HA protected cells lead to increased cell viability. It is concluded that the developed bioink has the potential to heal bone defects (Bendtsen et al., 2017). ECM derived materials and bioinks were also commonly used for bioprinting of bone tissues. For instance, ECM-derived HA was methacrylated and bioprinted by Poldervaart et al. In this work, photo-crosslinking of bioink was performed and human bone marrow-derived mesenchymal stromal cells (MSCs) were encapsulated. The fabricated construct had induced osteogenic differentiation, and MeHA bioink was appeared to be a promising bioink for fabrication of bone substitutes (Poldervaart et al., 2017). In a different report, Zhang and coworkers bioprinted alginate and gelatin by encapsulating human mesenchymal stem cells (hMSCs) into bioink to fabricate 3D bone-like tissue (Fig. 8.5A). They assessed the osteogenic differentiation of stiff (1.8% alginate) and soft (0.8% alginate) scaffolds and found that increased concentration of alginate makes the scaffold softer and induces osteogenic differentiation. The soft scaffold also provided increased cellular viability and these features made it a suitable bioink for bioprinting of cell-scaffold complexes for bone tissue engineering applications (Zhang et al., 2020).

8.4.2 Cartilage tissue engineering Cartilage tissue is a type of connective tissue that does not have self-renewal ability due to lack of blood vessels and limited cellular density. Therefore any

Table 8.2 Hydrogel bioinks for tissue engineering applications.

Bone tissue engineering

Cartilage tissue engineering

Bioink material

Bioprinting strategy

Gelation mechanism

Cell line



Chitosan Nanostructured Hydroxyapatite (HA)

Extrusionbased bioprinting

Temperature dependent gelation

MC3T3-E1 preosteoblast cells

Demirtas, ¸ Irmak, and ˘ Gümüsderelio ¸ glu (2017)

Alginate Polyvinyl alcohol HA

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Mouse calvaria 3T3-E1 (MC3T3) cells

Methacrylated hyaluronic acid (MeHA) Alginate gelatin

Extrusionbased bioprinting Extrusionbased bioprinting

Photo-crosslinking (Irgacure 2959)

Human bone marrow derived mesenchymal stromal cells (MSCs) Human mesenchymal stem cells (hMSCs)

• Induce mineralization • Good mechanical property • Improve cell viability • High shape fidelity • High cell viability • Induce bone defect healing • Induce osteogenic differentiation • Induce osteogenic differentiation • Increase cell viability

Zhang et al. (2020)

Nanocellulose Alginate

Extrusionbased bioprinting

Markstedt et al. (2015)

Collagen Agarose Alginate

Extrusionbased bioprinting


Primary chondrocytes from articular cartilage of newborn Sprague Dawley


Extrusionbased bioprinting

Air drying Warm drying Vacuum drying (NaOH)

ATDC5 (Mouse teratocarcinoma cells)

• Improve shear thinning properties • Provide shape fidelity • Printable at room temperature and low pressure • Improve mechanical strength • Increase cell proliferation • Proof of concept

Temperature dependent gelation and ionic crosslinking (CaCl2) CaCl2

Human nasoseptal chondrocytes (hNC)

Bendtsen, Quinnell, and Wei (2017) Poldervaart et al. (2017)

Yang et al. (2018)

Sadeghianmaryan et al. (2020)


Table 8.2 Hydrogel bioinks for tissue engineering applications. Continued

Cardiac tissue engineering

Bioink material

Bioprinting strategy

Gelation mechanism

Cell line



Nanofibrillated cellulose Alginate (A)

Extrusionbased bioprinting


Human nasal chondrocytes

Ávila et al. (2016)

GelMA Fibronectin Laminin Collagen methacrylate (ColMA) Alginate PEG Fibrinogen (PF)

Extrusionbased bioprinting


Human induced pluripotent stem cell

• Facilitates the biofabrication of patientspecific constructs • Provide homogenous cell distribution • Holds excellent shape postprinting • Leads to redifferentiation of hNCs • Induce the differentiation of hiPSCs into cardiomyocytes • Increase cell viability

Extrusionbased bioprinting

CaCl2 ionic crosslinking Photo-crosslinking

• Perfect shape fidelity • Repeatable construction • High precision

Maiullari et al. (2018)

Fibrinogen Gelatin HA

Extrusionbased bioprinting

Human umbilical vein endothelial cells (HUVECs) and iPSC-derived cardiomyocytes Primary cardiomyocytes from infant rat hearts

• Provide uniformity • Provide contractile and beating features • High electrical conductivity

Wang, Lee, Cheng, Yoo, and Atala (2018)

HA Alginate


Human embryonic kidney 293 (HEK-293) cells

Kupfer et al. (2020)

Rastin et al. (2020)

Neural tissue

Skin tissue engineering

Ti3C2 MXene nanosheets

Methylcellulose Alginate Gelatin Chitosan

Vascular tissue engineering

Polyurethane HA Glycerol Gelatin Fibrinogen GelMA PEGDA Nanosilicates GelMA Sodium alginate 4-arm poly (ethylene glycol)-tetraacrylate

Extrusionbased bioprinter

Extrusionbased bioprinting Extrusionbased bioprinting

Gallium nitrate

Human foreskin fibroblast cells

pH-dependent crosslinking (NaOH)

Human foreskin fibroblast cells

Extrusionbased bioprinting

Thrombin solution

Extrusionbased bioprinting Coaxial extrusion bioprinting

• • • • • • •

Low toxicity High biodegradability Excellent printability High shape fidelity High resolution Antibacterial features High cell viability

Rastin et al. (2021) Ng et al. (2016)

Human epidermal keratinocytes and human dermal fibroblasts

• Multilayered skin structure • High shape fidelity • High resolution • Provide a functional and quick reconstruction of facial skin


Human umbilical vein ECs, human umbilical artery smooth muscle cells

• Provide shear thinning property • Increase cell viability

Gold et al. (2021)

CaCl2 UV

HUVECs, human MSCs

• Support the spreading and proliferation of the cells • Tunable mechanical properties • Potentials in engineering large-scale vascularized tissue constructs

Jia et al. (2016)

Seol et al. (2018)


CHAPTER 8 Bioprinting of hydrogels

damage on it leads to a chronic condition and reduces the life quality of the patient. Autologous chondrocyte implantation, microfracture, and osteochondral grafts are current treatments for cartilage tissue degeneration (Singh, Bandyopadhyay, & Mandal, 2019). Although these treatments provide some improvements for regeneration, they still cannot ensure a complete recovery. There is a need for alternative and promising treatments. Emerging of 3D bioprinting technologies paved a way for creating designed cartilage tissue with proper features. Avila et al. used cell-laden nanofibrillated cellulose and alginate bioink to fabricate artificial auricular constructs (Fig. 8.5B). Results such as providing homogeneous cell distribution, maintaining post printing shape and leading to redifferentiation human nasal chondrocytes confirmed that developed hybrid bioink is promising for auricular cartilage tissue engineering and many other bio´ vila et al., 2016). Markstedt and coworkers, combined medical applications (A nanocellulose and alginate to develop a novel bioink that has a potential to be used in cartilage tissue engineering. In addition to fast crosslinking ability of alginate, nanofibrillated cellulose improved shear-thinning properties that provide shape fidelity while printing. As a result, a novel bioink that is printable at room temperature and low pressure was developed. In this study, optimal ratio of the nanocellulose and alginate was indicated as 80:20 (w/w) based on rheological properties and compression tests, as well as shape fidelity. This bioink formulation was used for bioprinting of chondrocytes, and cytotoxicity results showed that the developed bioink is highly biocompatible and suitable for cartilage tissue engineering (Markstedt et al., 2015). In another study, Yang et al. developed two types of composite hydrogel bioinks with the aim of using them in cartilage tissue engineering. These bioinks consisted of agarose and collagen, and they were used with the purpose of improving mechanical properties of alginate hydrogel. According to characterization results of the bioprinted scaffolds, both polymers showed improved mechanical properties. Also, collagen in the hydrogel provided additional benefits, such as better cell adhesion and increased cell proliferation. In addition to the increase in expression of cartilage specific genes (Acan, Sox9 and Col2a1), it decreased expression of Col1a1 gene, which is responsible for de-differentiation of chondrocytes. This ensures maintaining the phenotypes of cartilage. Due to its bioactivity and mechanical strength, the developed collagenalginate hydrogel was considered as a good bioink candidate for cartilage tissue engineering application (Yang et al., 2018). In the study of Sadeghianmaryan and coworkers, three different drying techniques; air drying, warm drying, and vacuum drying, were used to investigate the effect of crosslinking on mechanical properties of bioprinted hydrogel. Firstly, hydrogel concentration was optimized according to rheological properties and viscosity of varied chitosan hydrogel concentration. Discontinuous flow has been observed at lower concentration while clogging occurred at higher concentration. Mechanical properties are determined by elastic modulus. Based on the mechanical test results, air-dried hydrogel scaffolds had highest elastic modulus for minimum pore size. Also, interconnectivity of pores within air-dried scaffolds was confirmed by SEM analysis and high cell

8.4 Applications of hydrogel bioinks

viability was observed proving the biocompatibility of the scaffolds. According to all aforementioned results, 3D bioprinted chitosan hydrogel scaffolds that were fabricated by air drying method appeared to be the most promising construct for cartilage tissue engineering (Sadeghianmaryan et al., 2020).

8.4.3 Cardiac tissue engineering Creating a fully functional cardiac tissue or complex structure of artificial heart, which has the ability to pump blood is challenging. However, cardiac tissue engineering is progressing recently in terms of modeling cardiac tissue. Cardiac tissue models have started to replace animal models for the discovery of therapeutics. Although 3D bioprinting has limited studies in cardiac tissue engineering, it takes precedence over conventional and advanced manufacturing techniques with higher control during the scaffold production. Kupfer and coworkers tried to develop a bioink that induces the differentiation of hiPSCs into cardiomyocytes and provides high cell viability. Bioink consists of GelMA, ColMA, and noncrosslinked ECM proteins such as laminin and fibronectin. Results proved that ECM proteins included in bioink maintain stem cell proliferation and trigger differentiation. Long-term culturing resulted in the formation of contiguous muscle wall up to 500 μm thickness and the developed bioink was proved to be a suitable candidate for cardiac tissue engineering applications (Kupfer et al., 2020). In another study reported by Maiullari and coworkers, a bioink formulation composed of alginate (ALG, 4%) and polyethylene glycol-fibrinogen (PF, 1% w/w) was used for bioprinting of HUVECs and iPSCs derived cardiomyocytes. Crosslinking of ALG was performed by using CaCl2 solution, while photo-crosslinking was utilized to crosslink PF. Results showed that the developed bioink provided perfect shape fidelity and created myocardial-like tissue structures with repeatable construction and high precision (Maiullari et al., 2018). In another report, Wang et al. developed a contractile cardiac tissue by 3D bioprinting of fibrin-based composite hydrogel bioink that contains fibrinogen, gelatin, and HA dissolved in DMEM. They also used a sacrificial hydrogel and a supporting polymeric frame for bioprinting of a simple aligned cardiac construct (Fig. 8.5D). All bioprinting processes were carried out at 18 C to allow gelation of gelatin. After 3 days from bioprinting, the contractile feature of the cardiac tissue construct was assessed, the synchronous beating was observed starting from the first week, and until the third week contraction force was measured to be about 7.26 mN with the addition of epinephrine. This study has concluded that cardiac tissue models could be created by 3D bioprinting, since it provides cellular organization, uniformity, improved contractile, and beating features than scaffolds fabricated by other methodologies (Wang et al., 2018).

8.4.4 Skin tissue engineering Skin consists of three main layers which are epidermis, dermis, and hypodermis. These layers include different cells and ECM components that are responsible for



CHAPTER 8 Bioprinting of hydrogels

different functions. Obtaining multilayered structure of skin is the main goal of tissue engineering to mimic native skin tissue properly (Vig et al., 2017). Seol et al. used a composite material that consisted of PU, keratinocytes laden hydrogel, and fibroblast laden hydrogel to fabricate a face mask as a skin substitute (Fig. 8.5C). Result of this proof of concept study shows that bioprinted facial skin masks provided functional and quick wound healing and reconstruction of skin (Seol et al., 2018). Collagen is the most abundant protein that is included in the native ECM structure (Shoulders & Raines, 2009). Yet, its poor printability and requirement for toxic solvents are the common challenges of this biomaterial (Diamantides et al., 2017). Ng and coworkers used gelatin that is denatured version of collagen to develop native skin mimetic scaffolds. In this study, negatively charged gelatin was blended with positively charged chitosan polymer to obtain polyelectrolyte-complex hydrogel. Chitosan promoted cell attachment on the scaffolds due to the excessive positive charges, as well as the contribution on the gelation process. As a result, a 3-layered construct (approximately 400 μm) was bioprinted with high resolution and fidelity. This thickness was considered enough to mimic full of the epidermis and a part of the dermis. In addition, cytocompatibility results confirmed that the utilization of gelatin-chitosan hydrogel scaffold in skin tissue engineering applications is convenient (Ng et al., 2016). In addition to skin tissue engineering, bioprinted hydrogels are also used for contributing wound-healing applications. Bacterial infections are the most common and important challenges for wound-healing processes. To eliminate bacterial infection risks, antibacterial tissue scaffolds (Zakeri-Siavashani et al., 2020) and wound dressings (Simo˜es et al., 2018) have been started to be used. Rastin and coworkers developed a novel cell-laden bioink that consists of methylcellulose and alginate hydrogels. In this study researchers used gallium ion as a crosslinker, where gallium ions form ionic interactions between alginate chains to form hydrogel structure. In addition to its crosslinker role, gallium has an antibacterial feature. Therefore, it is highlighted as the key component of this study. Human foreskin fibroblast cell-laden methylcellulose alginate hydrogels were bioprinted and crosslinked in the gallium nitrate solution to produce antibacterial scaffolds. When cytocompatibility results were examined, cell viability of gallium crosslinked scaffolds was very close to cell viability results of calcium crosslinked samples. Considering all characterization results, antibacterial cell-laden methylcellulose alginate bioink was a promising candidate for skin tissue engineering due to its excellent printability, biocompatibility, and antibacterial potential (Rastin et al., 2021).

8.4.5 Vascular tissue engineering As the purpose of tissue engineering is to create a fully functional organ to replace the damaged or lost organs, vascularization has great importance. Creating an organ with the required vasculature could not be possible with conventional scaffold production methodologies (Huang, Li, et al., 2017; Huang,

8.4 Applications of hydrogel bioinks

Zhang, Gao, Yonezawa, & Cui, 2017). In recent years, 3D bioprinting has advanced in vascular tissue engineering and generating vasculature for artificial organs. Gold et al., developed a novel hydrogel-based bioink which consists of GelMA, PEGDA, and nanosilicates. Here, nanosilicates provided electrostatic interactions, and hydrogen bonds were formed between polymer chains and nanosilicates, which resulted in colloidal structure. Generally, GelMA and PEDGA have limitations for 3D printing methodology because of their Newtonian characteristics. In this case, addition of nanosilicates brought shear thinning property and improved the printability of the bioink. In addition, acquired shear thinning behavior prevented damaging of encapsulated human umbilical vein ECs (HUVECs) and human umbilical artery smooth muscle cells (HUASMC) in the bioink during the printing process. Cell viability results indicated that bioprinted cells keep their healthy phenotype and stay viable for approximately 1 month after bioprinting process. The reported cell-laden bioink was able to be bioprinted as anatomically accurate, and results confirmed that bioprinted vascular structure mimicked native human vascular tissue, properly. In conclusion, the bioprinted vascular mimetic structure has a potential to be used as 3D vascular disease model for drug development and screening applications (Gold et al., 2021).

8.4.6 Neural tissue engineering Neurodegenerative disorders, brain, and spinal cord damage are frequently encountered health problems, and modeling these diseases is crucial for understanding their mechanism and providing the required treatment. Bioprinting of hydrogel bioinks is used for modeling spinal cord injury and regenerating the spinal cord as illustrated in Fig. 8.5E (Joung et al., 2018). In addition to this, researchers have tried to develop conductive hydrogel bioinks for varied neural tissue engineering applications (Bordoni et al., 2020; Rastin et al., 2020; Vijayavenkataraman, Vialli, Fuh, & Lu, 2019). Although conductivity is one of the critical features of scaffolds that are used in neural tissue engineering applications, hydrogel bioinks are generally not conductive. Addition of some biomaterials, such as metal nanoparticles, carbon-based nanomaterials, or conductive polymers to the hydrogel bioinks provides electrical conductivity to bioink. Rastin and coworkers developed a novel cell-laden electroconductive bioink for neural tissue engineering applications. Addition of Ti3C2 MXene nanosheets provided electrical conductivity to cell-laden bioink that consists of HA and alginate. MXenes are novel metal carbide nanomaterials that possess large specific surface area, high electrical conductivity, low toxicity, and biodegradability features. Due to these excellent features, they are promising materials for biomedical applications. In the study, Ti3C2 MXene nanosheets were preferred due to their ease of dispersion in aqueous hydrogel solution. According to characterization results, developed electroconductive bioink was electrically conductive besides its excellent printability with high fidelity and resolution. In addition, MXenes improved the mechanical features due to interactions between MXenes and polymers in the



CHAPTER 8 Bioprinting of hydrogels

hydrogel. Cell viability studies indicated that viability of encapsulated human embryonic kidney 293 (HEK-293) cells in the hydrogel bioink is similar to that of in vitro studies that were performed by seeding cell to the hydrogel scaffolds. Considering all results, this study confirmed that the developed bioprinted electroconductive scaffold is an excellent candidate for neural tissue engineering applications (Rastin et al., 2020).

8.4.7 Drug screening For decades, 2D cell culture and animal models have been used for drug screening applications despite their limitations. Although 2D methods are convenient, 2D cultures provide monolayer cell culture that exhibit different cell morphologies, polarity, receptor expression, cell-ECM and cellacell interactions, as well as different chemical and physical properties, when compared to what is observed in vivo (Ravi, Paramesh, Kaviya, Anuradha, & Solomon, 2015). In addition, animal testing has long been used in science to study complex biological phenomena that cannot be investigated using 2D cell cultures. However, it appeared that there are more differences between animal models and human patients because of the vast divergence between their physiologies and anatomies, which brings many uncontrollable variables in the animal experiments. Moreover, experimental complexity, ethical concerns, and high costs are other common issues and disadvantages of animal testing. Considering all these limitations, animal models and 2D cell culture systems are not considered to be as effective as once assumed anymore for disease modeling and drug research. On the contrary, 3D cell culture systems proved to be effectively usable for disease modeling due to their superior properties such as successfully mimicking the cell-cell and cell- ECM interactions and providing relevant drug response compared to in vivo conditions (Chenchula, Kumar, & Babu, 2019). There are conventional and advanced methodologies to produce 3D scaffolds. Many approaches, such as solvent casting with particulate leaching, thermally induced phase separation (TIPS), and freeze drying which can be used to fabricate scaffolds in tissue engineering are considered conventional. Recently, some advanced techniques, such as electrospinning, 3D bioprinting, and combination molding techniques, have been developed to fabricate scaffolds that can mimic the ECM (Zhao et al., 2018). 3D bioprinting method can be used to produce artificial tissues and disease models by fabricating scaffolds with controlled spatial heterogeneity of physical properties, cellular composition, and ECM structure. This methodology has potential to create artificial functional constructs for drug screening and toxicology research (Arslan-Yildiz et al., 2016). In literature, there are several drug screening applications (Table 8.3) that utilized 3D bioprinted constructs. For example, Zhao and coworkers developed a 3D tumor model by using 3D bioprinting methodology. In the study, HeLa cells were used to create a cervical tumor microenvironment and they are encapsulated in the hydrogel bioink that is composed of fibrinogen, alginate, and gelatin. Morphological analyses results indicated that HeLa cells in the bioprinted

Table 8.3 Hydrogels used as bioink for drug screening applications. Bioink material

Bioprinting strategy

Gelation mechanism

Cell line



Fibrinogen Alginate Gelatin Vitrogel

Extrusionbased bioprinting

Thrombin CaCl2

HeLa (cervical tumor cell line)


Zhao et al. (2014)

Extrusionbased bioprinting

Docetaxel Doxorubicin Erlotinib

Gebeyehu et al. (2021)

Fibrin Alginate Genipin

Extrusionbased bioprinting

Thrombin CaCl2 Chitosan

Lung cancer (NSCLC-PDX, H460, HCC827and A549) and breast cancer (MDAMB-231WT), and bladder cancer (RT4) cell lines U87MG human glioblastoma cells

Lee et al. (2019)

Alginate Gelatin Fibrinogen Thrombin Gelatin Fibrin Pluronic Alginate Pluronic

Extrusionbased bioprinting

Ionic crosslinking (CaCl2) Transglutaminase

Glioma stem cell line SU3 Human glioma cell line U87

Glioblastoma-reprogramming cocktail [Forskolin, ISX9, CHIR99021, I-BET 151, DAPT (FICBD)] Temozolomide

Extrusionbased bioprinting

Thrombin Transglutaminase

Proximal tubule epithelial cells

Resazurin, Cisplatin, Cyclosporine A

Homan et al. (2016)

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Murine C2C12 cells


Gelatin Alginate Matrigelt

Extrusionbased bioprinting

Ionic crosslinking (CaCl2)

Intrahepatic cholangiocarcinoma cells (ICC)

Sorafenib, Cisplatin, 5Fluorouracil

Mozetic, Giannitelli, Gori, Trombetta, and Rainer (2017) Mao, Yang et al. (2020), Mao, He et al. (2020)

Dai, Ma, Lan, and Xu (2016)


CHAPTER 8 Bioprinting of hydrogels

hydrogel constructs formed spheroid structures. Moreover, 3D bioprinted constructs allowed long-term cell proliferation due to increased cellcell and cellmatrix interactions like real tumor tissue. Furthermore, tumor-related protein expression levels in 3D constructs were found to be higher than 2D culture, and it confirmed that 3D bioprinted constructs successfully mimicked native tumorigenic tissue. To investigate drug resistance of HeLa cells in the 3D constructs Paclitaxel was preferred as an antitumor drug. When it was compared to 2D cell culture, 3D cervical tumor model was more resistant to Paclitaxel and it showed the importance of the dimensionality on drug screening studies (Zhao et al., 2014). Recently another approach reported by Gebeyehu et al., a commercial hydrogel (VitroGel) was modified with the aim of improving printability, rheological characteristics, and stability without requiring crosslinking strategies such as UV exposure, sudden changes in temperature, or chemical reactions. Thus, they overcame the challenges that caused cell death during bioprinting and solidification steps. Characterization results showed that RGD modified hydrogel had the best characteristics with high shape fidelity along with high cell viability. In this study, the efficacies of the docetaxel, doxorubicin, and erlotinib which are anticancer drugs were investigated on various cancer types by bioprinting 3D models of RGD modified hydrogel bioink. Drug screening results revealed that developed 3D bioprinted tumor models were more resistant than 2D cell culture models. According to these results, researchers concluded that the developed bioink was very convenient for 3D bioprinting process due to its rheological features and biocompatibility (Gebeyehu et al., 2021). In another work, Lee and coworkers proposed a novel fibrin-based 3D printed construct for modeling glioblastoma multiforme, which is one of the deadliest cancer types. In the study, a microfluidic print head was used to decrease the effect of shear stress which causes cell death during the bioprinting process. The microfluidic print head allows printing cell-laden bioink and crosslinker simultaneously. To create a glioblastoma multiforme model, U87MG (human glioblastoma) cells were encapsulated into bioink. Live dead assay images were taken shortly after the bioprinting process showed that the cell viability was quite high. These results proved that cells were not harmed by shear stress during the bioprinting process. Also, in the following days, single cells started to form spheroid structures that mimic native tumor structure, and high cell viability was maintained up to day 12. In addition, high expression levels of glioblastoma multiforme markers confirmed the convenience of 3D cancer model. Furthermore, developed cancer model was used for screening of novel glioblastoma multiforme treatment methods, which were only screened by 2D models previously. Drug screening results revealed that developed 3D cancer model was more resistant than 2D cell culture. These results supported that developed 3D printed construct mimicked native tumor structure and it was promising to model other complex diseases for drug development and screening applications (Lee et al., 2019).

8.5 Challenges of bioprinted hydrogels in tissue engineering

8.5 Challenges of bioprinted hydrogels in tissue engineering and drug screening Although there are several advancements for using 3D bioprinting in tissue engineering and drug screening, the development of a perfect bioink is still unmet. Features of a perfect bioink were discussed in detail at Section 8.2. Briefly, bioink materials should have fast gelling features, convenience for simple as well as nontoxic crosslinking mechanisms, and should be capable to be bioprinted with a low amount of pressure application to prevent cell death. Having a proper mechanical property regarding intended use, and good printability are also other features that a bioink should meet (Leberfinger et al., 2019). Hydrogels are commonly used and promising bioink materials in the field of tissue engineering. However, printability analyses of each hydrogel are yet to be performed. As listed in Table 8.2 and Table 8.3, gelatin, GelMA, alginate, and chitosan are the most preferred hydrogels for 3D bioprinting. In literature, many printability studies and their bioprinting parameters are readily optimized for above mentioned hydrogels and their derivatives. There are also various hydrogels whose bioprinting parameters need to be optimized for each bioprinting strategy. Printability of some hydrogels such as collagen is not possible at all without dissolving them in harsh and toxic solvents (Diamantides et al., 2017). Hence, cellular viability is an important challenge in such cases. Viscosity is another critical parameter, but can also be a challenge for bioprinting process. The rheological properties and viscosity of hydrogels have significant importance on printability and shape fidelity. If the viscosity of hydrogel is low, it can flow out from the cartridge; on the contrary, it can clog the tip of the extruder if the viscosity is too high. To prevent this, examination of the rheological properties and viscosity of hydrogels have great importance (Gao et al., 2018). Another challenge is decreasing of cellular viability based on high pressure applied during the bioprinting of cell-laden hydrogels. Extrusion of hydrogel from the cartridge requires application or generation of high pressure, the applied pressure cause stress on cells, and this results in reduced cellular viability. Therefore the ability of the hydrogel to be able to be extruded at low pressure is critical for cell-laden hydrogels (Nair et al., 2009). The mechanical strength of hydrogel bioinks is one of the challenging limitations especially for some specific tissue engineering applications. Regarding the intended use of scaffold, the hydrogel should meet some specific biomechanical properties. However, since hydrogels can retain water and are softer than most of the other synthetic polymeric materials, they are widely used for replacement of soft tissue. The stiffness of hydrogel can be achieved by increasing the crosslinking density and molecular weight (Huang, Li, et al., 2017; Huang, Zhang, et al., 2017). However, it can also result in decreased cellular viability because cells get entrapped into the polymer network, which limits diffusion of nutrients and oxygen (Chimene, Kaunas, & Gaharwar, 2020). To overcome the challenge of low



CHAPTER 8 Bioprinting of hydrogels

mechanical strength, composite bioinks can be preferred. Composites or polymer blends can provide mechanical stability while natural components of these blends provide a proper microenvironment for cell adherence and growth. This can be achieved either by bioprinting of blended composite scaffold or heterogeneous layer-by-layer printing (Mao, He, et al., 2020; Mao, Yang, et al., 2020). Resolution is one of the most important problems encountered in tissue and organ bioprinting with hydrogels. Although LAB has a higher resolution than extrusion-based bioprinting and droplet bioprinting, encapsulation of cells into hydrogel decreases the resolution and this prevents the bioprinting of fine filaments (Ozbolat & Yu, 2013). Despite the final construct having the desired shape, it cannot conserve the shape postprinting and can spread or shrink. To prevent this, the resolution of utilized strategy needs to be improved. Lastly, as in all tissue engineering methodologies, the inability of vascularization creates an important limitation. Maintaining the viability of bioprinted tissue or organ model cannot be possible without vascularization (Ozbolat & Yu, 2013). As a result, bioprinting of hydrogels for tissue engineering and drug screening purpose has still several challenges and limitations, which could be eliminated by further research and experimentation.

8.6 Conclusion and future perspectives Creating fully functional tissue constructs and artificial organ-like structures have great importance for tissue engineering and regenerative medicine applications, also modeling diseases would provide better opportunity to reduce the cost during drug discovery process. 3D bioprinting of hydrogels has numerous advantages for scaffold/tissue fabrication and has great potential in utilization for bone, cardiac, cartilage, neural, skin, vascular tissue engineering, and drug screening as given in Section 8.4. On the other hand, hard processibility of some hydrogels and lack of their optimized bioprinting parameters, low mechanical strength of hydrogel bioinks, low resolution in the case of cell-laden bioink, and the inability of vascularization limits the successful bioprinting of hydrogels for fully functional tissue constructs. Developing new generation bioinks with improved features and creating composite hydrogel scaffolds to provide better mechanical properties and increased cellular viability (Schwarz et al., 2020; You, Chen, Cooper, Chang, & Eames, 2018; Zehnder, Sarker, Boccaccini, & Detsch, 2015) would possibly eliminate the obstacles (Chimene, Lennox, Kaunas, & Gaharwar, 2016) that were explained in the previous section. With the advancements in both bioprinting technology and bioinks, it is expected that clinical applications of 3D bioprinted tissue/organ transplantation dream will come true. In addition, in terms of drug discovery and drug screening, it is anticipated to reduce the cost by creating disease models using the 3D bioprinting approach (Peng et al., 2017). As expected in the tissue engineering perspective,


developments in 3D bioprinting technology and hydrogels will pave the way for personalized medicine, reduce the cost for drug discovery. Besides, in recent years, bioinks have started to enter product portfolio of many companies, and it is foreseen that the new generation bioinks to be developed will take their place in the market as in great demand commercial product either for tissue engineering and regenerative medicine studies or transplantation (Guvendiren, 2019; Roskos, Stuiver, Pentoney, & Presnell, 2015).

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CHAPTER 8 Bioprinting of hydrogels

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Smart polymers for biomedical applications


Deepti Bharti1, Indranil Banerjee2, Preetam Sarkar3, Doman Kim4 and Kunal Pal1 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of Bioscience & Bioengineering, Indian Institute of Technology Jodhpur, Jodhpur, Rajasthan, India 3 Department of Food Process Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 4 Department of International Agricultural Technology and Institute of Green BioScience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea

9.1 Introduction The response toward any external stimulus as a mechanism to adapt to the changing environments is an existing phenomenon for most living systems. With the exact mindset, many polymer scientists are working progressively to search and develop a polymeric system that can behave similarly to the living system. The search has landed researchers in a particular class of polymers with unique chemical and physical properties, which has found applications in various biomedical fields like drug delivery and tissue engineering. These polymers are commonly associated with names like “environment-sensitive polymer,” “smart-polymer,” “stimuli-responsive polymer,” and “intelligent polymers” (Kumar, Srivastava, Galaev, & Mattiasson, 2007). These polymers will be further referred to as smart polymers (SP) for clarity. In simple terms, SPs have the potential to overcome and adapt to even trivial environmental changes. These environmental conditions are a set of thermodynamic constraints like pressure, temperature, pH, and concentration (Galaev & Mattiasson, 2019). Therefore SPs are thermodynamic systems that undergo reversible changes through changes in the chemical structure of phases inside the range of different environmental constraints. The system of SP is exclusive not because of its ability to produce rapid macroscopic changes but the reversible nature of these transitions. Hence, scientists have a vast opportunity to explore polymer chemical structure, compositions, architectures, and environmental conditions before designing a suitable SP for any application. The solubility, degradation, structural and molecular rearrangement, along with the balance of lipophilic and hydrophilic groups of the polymers, are a few more essential considerable information (Ribeiro & Flores-Sahagun, 2019). SP responds to either Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 9 Smart polymers for biomedical applications

single or multiple external stimuli like pH, temperature, biological molecules, light, etc. The variation in the polymers and their structural conformations can either occur gradually or abruptly. The response of the polymers against the different stimuli is observed in terms of variation in the macromolecular structure, swelling characteristics, structural collapsing, or simply a solution to gel transition (Aguilar & Roma´n, 2014). The transition from sol to gel is attributed to the hydrophilic group of the polymer, which adsorbs water. It can be seen that the response of SPs is always an effort to correlate with the living systems. This is further supported by the evidence that most polymers have a transitional temperature of B37 C (Galaev &. Mattiasson, 2019). It is the physiological temperature where many biological molecules like proteins, polysaccharides, and nucleotides function typically. The environmental stimuli can be classified majorly into three groups: physical stimuli, chemical stimuli, and biological stimuli, which is also the basis for categorizing SPs (Ribeiro & Flores-Sahagun, 2019). Any physical stimuli will lead to changes in the molecular and structural dynamics of the polymeric system. Changes in response to temperature, pH, magnetic and electric fields, and light are a few examples of physical stimuli. The chemical stimuli will alter the molecular interaction between the polymeric chains or the chain and solvent system. An alteration in the concentration or ionic strength can be considered as chemical stimuli. The physical and chemical stimuli-based SPs are often associated with chemical contamination, cell damage, and low biological specificity (Xu, Liu, & Yan, 2017). A relatively new area of research is with the biological stimuli that will improvise the enzymatic reactions that might help recognize the targeted cell receptors. Sometimes SPs are even designed to understand the influence of more than one physiological or environmental stimuli. Conjugating many natural and synthetic biomolecules often design the SPs to their backbone (Hoffman & Stayton, 2020). These conjugations usually act as targeting signaling molecules or simply as a key for cellular entry. All the environmental alterations mentioned above can be accounted for developing SPs for drug delivery systems and, in fact, substrates for culturing cells for tissue regeneration. Fig. 9.1 represents different stimuli that are often explored for the development of SPs. One of the most explored applications of SPs has been observed in the case of drug delivery. The SP-based micro-/nanocarriers that encapsulate drugs/biologics are used for controlled or targeted delivery (Xu et al., 2017). For drug delivery, many formulations constituting SPs like Bion Tears, Polygel, and Genteal Gel, are already in the market with appreciable performance (Thrimawithana, Rupenthal, Young, & Alany, 2012). Many researchers have recently explored SP to design scaffolds for tissue regeneration in bone, cartilage, etc. (Haryanto & Khan, 2021). SP formulated as gels can also be implanted at the damaged tissue site in a minimally invasive manner. This chapter has made an effort to guide the readers through a complete understanding of the functioning of SP. The main aim will be to summarize the conceptual working of the various environmental stimuli attributed to the polymer’s “smart” behavior. The chapter will cover a wide range of stimuli, that is,

9.2 Temperature-sensitive smart polymers

FIGURE 9.1 Various stimuli for the development of smart polymers.

temperature, pH, light, enzyme, and their specific role in regard to the SP. Additionally, it will cover the recent advancements of the applications of SPs in biomedical, especially drug delivery, tissue repair, and regeneration.

9.2 Temperature-sensitive smart polymers Temperature-sensitive smart polymers (TSSPs) became a topic of discussion in the late 1960s after the first report of thermal phase transition in poly(N-isopropyl acrylamide) (pNIPAAm) (Heskins & Guillet, 1968). Since then, TSSPs have been most explored by polymer scientists, particularly in the field of biomedicine. At a critical solution temperature (CST), these SPs display sensitive behavior through changes in their structural and functional properties. Based upon the response of the polymers, CST may be defined either as lower critical solution temperature (LCST) and upper critical solution temperature (UCST) (Teotia, Sami, & Kumar, 2015). The phase diagram of polymer weight fraction versus temperature (Fig. 9.2) depicts mono-phase and biphase regions which are helpful in the identification of LCST and UCST (Gibson & O’Reilly, 2013). The temperature at



CHAPTER 9 Smart polymers for biomedical applications

FIGURE 9.2 Schematics of the phase diagram of polymer solution: (A) lower critical solution temperature and (B) upper critical solution temperature. Reproduced from Gibson, M. I., & O’Reilly, R. K. (2013). To aggregate, or not to aggregate? considerations in the design and application of polymeric thermally-responsive nanoparticles. Chemical Society Reviews, 42(17), 7204 7213. under Creative Commons license.

which the phase separation takes place is called cloud point, a quantifiable macroscopic event of the transition process (Gibson & O’Reilly, 2013). The LCST depicts the lowest temperature of the phase diagram below which the polymers in aqueous solvent remain monophasic (soluble). On the contrary, the polymeric system displaying single-phase above a specific temperature is classified under UCST. Similar to any process to become thermodynamically stable, the polymer dissolution also requires a negative value for Gibbs free energy (ΔG) (Teotia et al., 2015). For an overall negative value of ΔG (ΔG 5 ΔH 2 TΔS), the term of enthalpy (ΔH) and entropy (ΔS) should also be negative. The negative value of ΔH assists in polymer dissolution and is related to the hydrogen bonding between the polymer and the solvent (Pattanashetti, Heggannavar, & Kariduraganavar, 2017). Similarly, the negative ΔS value comes from the organized arrangement of water molecules around the polymer. A rise in the temperature beyond LCST results in the system’s increased entropy. Hence, the overall value of ΔS exceeds ΔH that leads to the breakage of hydrogen bonds and further separation of phases. In comparison to UCST, the LCST polymers are explored by scientists for the majority of the applications. pNIPAAm, poly(N,N-diethyl acrylamide), poly(N-vinyl alkylamine), and poly(N-vinyl caprolactam) are a few of the commonly used thermo-sensitive polymers (Aguilar & Roma´n, 2014).

9.3 Applications of temperature-sensitive smart polymers The biomedical applications of temperature-sensitive SPs are well explored in drug delivery, tissue engineering, and regenerative medicines. Among various SPs

9.3 Applications of temperature-sensitive smart polymers

under this category, pNIPAAm is majorly explored due to its solubility in aqueous solutions and LCST of 32 C (Mu & Ebara, 2020). The sensitivity of this polymer toward the CST can be correlated to its structure which is made from amide linkage (hydrophilic) and isopropyl portion (hydrophobic) represented in Fig. 9.3 (Yang, Fan, Zhang, & Ju, 2020). The synthesis and functionalization of pNIPAAm occur through the free radical polymerization of monomers. One of the beneficial roles of pNIPAAm occurs in culturing of the mammalian cell. This specific application is required to recover cells from the surface to which they adhere for growth and proliferation. Since the techniques of mechanical scraping or enzymatic treatments may prove harsh for the cultured cells, the development of temperature-sensitive substrates might serve a beneficial role here. The vapor phase deposition of pNIPAAm on polystyrene-coated plate showed adherence of most cell types above the LCST for proper growth and proliferation. However, below the LCST the surface allows the cells to detach due to the hydration of pNIPAAm coating (Canavan, Cheng, Graham, Ratner, & Castner, 2004). The immunological assay revealed that proteins like fibronectin, laminin, and collagen were found to be closely associated with the detached cells. However, some collagen is allied with the surface.

FIGURE 9.3 (A) The molecular structure and mechanism of thermo-sensitivity of pNIPAAm. (B) The schematic representation of thermo-responsive behavior of the stimuli-responsive hydrogel-based drug delivery system. (A) Reproduced from Yang, L., Fan, X., Zhang, J., & Ju, J. (2020). Preparation and characterization of thermoresponsive poly(N-isopropylacrylamide) for cell culture applications. Polymers, 12(2), 389. https://doi. org/10.3390/POLYM12020389 under Creative Commons license. (B) Reproduced from Li, L., He, Y., Zheng, X., Yi, L., Nian, W., & Abadi, P. P. (2021). Progress on preparation of pH/temperature-sensitive intelligent hydrogels and applications in target transport and controlled release of drugs. International Journal of Polymer Science, 2021. under Creative Commons license.



CHAPTER 9 Smart polymers for biomedical applications

The thermoresponsive behavior of SP for drug delivery can be understood through Fig. 9.3B (Li et al., 2021). The closeness of LCST of pNIPAAms to physiological temperature makes them suitable for drug delivery applications. Alginate-g-pNIPAAm copolymers have been studied in this regard for the controlled release of hydrophobic anticancer drugs (Liu, Song, Wen, Zhu, & Li, 2017). The clear idea here is that the temperature-sensitive hydrogel will yield self-assembled micellar structures at 37 C, followed by the dissolution of the polymer in the biological fluid. Thus the drug was released in an encapsulated form in the micelles formed by the polymers, which even showed enhanced cellular uptake when tested in vitro in multidrug-resistant cells. Chitosan is a natural polymer derived from the deacetylation of chitin. It is often united with polymers holding thermoresponsive characteristics for the synthesis SP. Carboxymethyl chitosan (CMCh) modified pluronic gels were studied for localized delivery of paclitaxel with reported LCST of B29 C (Ju, Sun, Zi, Jin, & Zhang, 2013). In the fabricated pluronic gel system, glutaraldehyde-mediated cross-linking took place to generate a stable network with better mechanical strength. The formed hydrogel displayed excellent drug holding ability, sustained release, reduction in hepatic metabolism, and improved anticancer ability. Another application of thermo-sensitive SP is seen in gene therapy. Gene therapy serves as a corrective treatment for defective genes, which can potentially cause different genetic diseases. The significant role will involve the delivery of the therapeutic gene (deoxyribonucleic acid, DNA) to the damaged site, where repair and replacement will occur. The negative charge of the DNA will face hindrance from the negatively charged cellular membrane before it finally reaches the nucleus. Accordingly, the gene needs to be packed in a suitable carrier/vector for its delivery. For most gene-targeted diseases, the temperature is considered a factor for the successful release of the gene from the carrier (Liu, Yang, Xiong, & Gu, 2016). A thermoresponsive nanogel was prepared through radical graft copolymerization of pNIPAAm to PEI. The nanogels were found effective in anticancer gene therapy (Cao et al., 2015). PEI is a low immunogenic polymer. Its cationic nature can assist in condensing DNA molecules in small size, which assists in endocytosis. pNIPAAm enhances the cellular uptake when the temperature rises the LCST. A tumor suppressor gene, that is p53, was efficiently loaded and delivered through the synthesized temperature-responsive PEI/pNIPAAm (Cao et al., 2015). A list of thermoresponsive polymers and their potential applications is represented (Table 9.1).

9.4 pH-sensitive smart polymers The different human tissues and their anatomical location exhibit a variation in the pH. The SPs which respond to the change in the variations of environmental pH are categorized as pH-sensitive SP. These responses occur in terms of

9.4 pH-sensitive smart polymers

Table 9.1 List of few thermoresponsive polymers and their applications. S. No.


Polymer type

Therapeutic agent


Cytosinepolypropylene glycol

LCST (35 C)




LCST (35 C)




LCST (37 C)


Chitosan/ glycerophosphate

LCST (37 C)

Codelivery of Dox, cisplatin, and methotrexate Chondrocytes



Doxorubicin and vaccinia virus vaccine



Efficient for drug loading and sustained release when tested for small cell lung cancer cell line Complete inhibition of tumor growth in vivo in mice having a drugresistant tumor Inhibition of tumor growth and tumor necrosis Capable of maintaining high cell viability and expression of cartilagespecific genes Displayed a synergistic antitumor effect when combined with the vaccinia virus

Cheng, Liang, Liao, Huang, and Lee (2017)

Fan et al. (2018)

Ma et al. (2015)

Zhang et al. (2019)

Han et al. (2008)

LCST, Lower critical solution temperature; PLGA, poly(lactic-co-glycolic acid); pNIPAAm, poly(Nisopropyl acrylamide), PEG, polyethylglycol.

modification to the different properties of the polymers like structure, solubility, surface property, etc. (Ofridam et al., 2021). The fundamental structure of a pHsensitive polymer includes an ionizable group that is connected to the hydrophobic polymeric backbone. The pH-sensitive SPs are similar to polyelectrolytes and consist of acidic or basic groups. These acidic or basic functional groups respond to the external environment by accepting or donating the protons, respectively. The elementary categorization of pH-sensitive SPs is done based on the surface charge possessed by these polymers. Broadly, they are categorized into two groups: polyacid polymers consisting of anionic functional groups, and polybasic polymers consisting of cationic functional groups. Some of the common examples of polyacid polymers include poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMA). In contrast, the polymers like PEI and poly



CHAPTER 9 Smart polymers for biomedical applications

(dimethylaminoethyl methacrylate) are categorized as polybasic polymers (Roy & De, 2014). Polyacid polymers consist of an acidic group like carboxylic acids and sulfonic acids. At high and neutral pH values, they tend to lose protons, thereby forming a negatively charged polymer chain; however, at low pH, it accepts protons (Kocak, Tuncer, & Bu¨tu¨n, 2016). On the contrary, polybasic polymers are enriched with basic functional groups like amines and imidazole. They form positively charged polymer at low pH by accepting the protons (Kocak et al., 2016). Therefore the protonation and deprotonation of these groups tune the net charge on the polymers (Bazban-Shotorbani et al., 2017). The increase in overall net charge causes a transition of the chains from a collapsed state to an expanded state. On the other hand, a decrease in the net charge will cause a change to a collapsed state. The pH-sensitive polymers are explored for a wide range of applications because of their biocompatibility. Eudragit L and Eudragit S from Ro¨hm Pharma GmBH, and CMEC (derived from cellulose) produced by Freund Sangyo Co., Ltd. are few commercialized pH-sensitive polymers (Aguilar & Roma´n, 2014). This pH sensitivity depends on the structure of the ionizable groups and other environmental factors like polymer composition, ionic strength, and properties of the polymeric backbone (Moo Huh, Kang, Lee, & Bae, 2012). In response to a change in the environmental pH, the net charge of polymeric structures like hydrogels typically displays swelling or deswelling behavior. The changes mentioned above occur at specific pH for a specific polymer, thus called critical pH (pH ) (Bazban-Shotorbani et al., 2017). pH depends upon the pKa value, which is defined as the pH at which half of the ionizable groups of polyelectrolytes are ionized (Bazban-Shotorbani et al., 2017). Selecting a polymer with the desired pH based upon the environmental condition is crucial in developing these polymers. Few of the early studies on pH-sensitive polymers were focused on the delivery of bioactive agents to the gastrointestinal (GI) tract, which depicts a great fluctuation in the pH. The copolymerization method allows the designing of polymers with a desired pH . Additionally, hydrophobic modification of polyelectrolytes can also be used to shift the pH value. Thus a complete understanding of the polymers’ ionizable group, their chemical structure, and an estimation of their pKa value is required for the development of pH-sensitive polymers.

9.4.1 Applications Researchers have explored the existence of pH variations within the human body to design smart delivery systems. The topical or transdermal route for drug delivery is considered a safe and patient-friendly way to deliver drugs to the human body. The pH of human skin is around 5; however, in melanoma, the pH varies from 5.5 to 7. A pH-responsive poly(lactic-co-glycolic acid) (PLGA)/chitosan nanogel was synthesized to achieve sustained delivery of anticancer drug 5-fluorouracil to the dermal tissue (Sahu, Kashaw, Jain, Sau,

9.4 pH-sensitive smart polymers

& Iyer, 2017). The drug was incorporated in the PLGA matrix by “solvent evaporation emulsification” followed by layering a chitosan coat (Sahu et al., 2017). The drug release pattern showed a sustained release due to PLGA particles. Further, pH-sensitive behavior and site-specific drug release characteristics of the formulations were revealed through the study. The formulated gel improved the penetration of the drug into the stratum corneum. The developed formulation was found to be a suitable candidate for transdermal drug delivery against skin cancer. Delivery of drugs through the oral route is also considered a convenient approach for the administration of drugs. However, there is a possibility that drugs administrated through the oral route might face some hindrance at the later stage when it reaches to GI tract. Additionally, many therapeutic peptides and proteins will also prove to be unstable under the stomach’s acidic environment. Hence, pH-sensitive oral delivery systems can be designed to protect the therapeutic peptides from the harsh acidic environment. One such example is the use of pH-sensitive alginate nanogels for the oral delivery of peptides. In this regard, alginate nanogel was developed using a microfluidic platform (Bazban-Shotorbani et al., 2016). Alginate nanogel behaves as an anionic polyelectrolyte, and its swelling is dependent upon its degree of ionization. The proposed system was found to be a promising approach for the encapsulation of polypeptides and their sustained release. Silica nanoparticles (SNPs) are an excellent drug carrier because of their large specific surface area, better mechanical stability, and low cell toxicity. A pH-sensitive polymer poly(2(diethylamino)ethyl methacrylate) (PDEAEMA), when grafted with SNP, has shown an improvisation of the therapeutic efficacy (Fig. 9.4) (Xu, Li, & Wang, 2019). This polymeric system was explored to deliver quercetin, whose anticancerous application is often limited due to its poor bioavailability. The formulated delivery system remained compact under the physiological pH (pH 5 7.4) and did not release the entrapped drug molecules. However, when the pH turns slightly acidic (pH 5 5.5), the system disintegrates because of the amine groups’ protonation present in PDEAEMA (Xu et al., 2019). This results in the release of quercetin from the polymer micelle. The pH variation at the wound site is also dynamic. However, this variation in the pH is often not given due diligence while designing a wound dressing material. A smart dressing material can be created using pH-sensitive polymers, acting in accordance to wound healing. The cationic nature of chitosan has been revealed to be constructive in this regard. Chitosan has a pKa value of 6.5, forming a stable gel at basic pHs and a swollen or dissolved polymer in the acidic environment (Chen et al., 2004). The swelling property of chitosan is beneficial in the initial stages of healing, where it improvises cell infiltration, proliferation, and oxygen permeability. Zhu and Bratlie (2018) have reported the synthesis of a pH-sensitive hydrogel using methacrylate chitosan. The fabricated gel released antiinflammatory factors during the initial stages of healing and further accelerated the healing process. pHsensitivity chitosan-based systems are beneficial in tissue repair and regeneration



CHAPTER 9 Smart polymers for biomedical applications

FIGURE 9.4 Synthesis and pH-dependent release from SNPs-g-PDEAEMA. Reproduced from Xu, L., Li, H.-L., & Wang, L.-P. (2019). pH-sensitive, polymer functionalized, nonporous silica nanoparticles for quercetin controlled release. Polymers, 11(12), 2026. POLYM11122026 under Creative Commons license.

applications. In this regard, self-assembled CMCh and amorphous calcium phosphate hydrogel served as an ideal scaffold material for bone tissue regeneration (Zhao et al., 2019). The formed hydrogel supported proper growth, proliferation, and differentiation of mesenchymal stem cells (Table 9.2).

9.5 Photosensitive polymers Photochemistry has a central role in many biological processes, including photosynthesis, circadian cycle regulation, and sight. Light as a stimulus has recently gained attention in the biomedical field, where researchers are trying to explore its potential to regulate biological functions. The possible reason can be the spatiotemporal localization of light and its ability to activate a system even from outside. The photosensitive polymers display a reversible nature in their chemical and structural properties when exposed to light. An adequately engineered photosensitive polymer with appropriate knowledge of photochemistry can widen the opportunities of altering biological processes with great precision. There are several advantages associated with using light as a stimulus. Firstly, light includes a wide range of available wavelengths ranging from ultraviolet to infrared; secondly, it provides a possible four-dimensional control over the material’s responsiveness. Thirdly, the control over the administered light dose under in vivo conditions assists in functional regulations (Ruskowitz & DeForest, 2018).

Table 9.2 pH-responsive polymers and their range of applications. Loaded therapeutics






Treatment of Helicobacter pylori infection



Treatment of H. Pylori infection


Slightly basic

Divinyl sulfone



Delivery at the lower (gastrointestinal) GI tract to treat inflammatory bowel disease and ulcerative colitis Inhibition of the growth of acne

El-Mahrouk, Aboul-Einien, and Makhlouf (2015) Risbud, Hardikar, Bhat, and Bhonde (2000) Das and Subuddhi (2015)



Gene therapy




Cross-linked with tripolyphosphate Cross-linked with glutaraldehyde Tetraethyl orthosilicate

Chitosan and poly(vinyl pyrrolidone) Acrylic acid grafted guar gum/β-cyclodextrin Hydroxyethylcellulose/ hyaluronic acid Poly(Nisopropylacrylamide)-copoly(acrylic acid)-co-poly (caprolactone)

Kwon, Kong, and Park (2015) Hu, Zhang, Zhang, Xu, and Zhuo (2009)


CHAPTER 9 Smart polymers for biomedical applications

These advantages altogether allow light-sensitive polymers for a wide variety of applications. Additionally, the photochemical reaction does not require extra reagents apart from the light stimulus, and they also have limited by-products (Bertrand & Gohy, 2016). The functional aspects of photosensitive polymers depend upon the extent and rate of change that occur due to light triggering and the reversibility of the process when the triggering light source is withdrawn (Cui & Del Campo, 2014). The fundamental structure of photosensitive polymer includes a photosensitive moiety and a bulk polymer (Upadhyay, Thomas, Tamrakar, & Kalarikkal, 2019). Photoresponsive polymers are synthesized by incorporating a chromophore, a light-sensitive functional group, in the polymer chain. The type of active group decides the reversibility of the process. Additionally, these groups are crucial in determining the dynamic behavior of the synthesized polymer. Azobenzene, spirooxazine, spiropyran, and fulgide derivatives are a few examples of chromophores (Schattling, Jochum, & Theato, 2013). These chromophores are capable of absorbing specific wavelengths of light. When the polymer solution is irradiated with light, the chromophores undergo photoinduced isomerization, dimerization, or cleavage, thus converting light into a chemical signal (Fig. 9.5; Ferna´ndez & Orozco, 2021). The photoisomerization process changes the molecular structure among the two isomers when kept under light irradiation (Upadhyay et al., 2019). Azobenzene and spiropyran are typical examples that let the polymer undergo photo-isomerization. It is a reversible process and results in changes in the polymer’s physical properties (e.g., color, refractive index, conductivity, etc.) (Ercole, Davis, & Evans, 2010). Photodimerization includes a chemical reaction between the photoexcited and an unexcited molecule within the same molecular species (Upadhyay et al., 2019). Like photo-isomerization, dimerization is also a reversible process and commonly incorporates photosensitive behavior in the polymer chain. Various molecules like cinnamylidene acetate and nitrocinnamate exhibit the photo-dimerization process (Zheng et al., 2001). Photocleavage, as the name suggests, involves the breaking of molecules under the influence of light. They comprise molecules that are unstable under the influence of light. Due to their said advantages, photoresponsive polymers have been explored in the biomedical field as photo-actuators, therapeutic hydrogels, optical devices, etc.

9.5.1 Applications Among the various applications of photosensitive polymers, their role in targeted in vivo drug delivery is appreciable. One example in this regard is the synthesis of polymers as photochromic vesicles using poly(ethylene oxide)-b-PSPA, in which SPA stands for spiropyran (Wei, Gao, Li, & Serpe, 2016). Inside the polymeric vesicle, there occurs a photoinduced isomerization between the spiropyran and merocyanine. This transition is helpful in tuning the permeability of the

9.5 Photosensitive polymers

FIGURE 9.5 Photoresponsive molecules for photo-triggered targeting. Reproduced from Ferna´ndez, M., & Orozco, J. (2021). Advances in functionalized photosensitive polymeric nanocarrier. Polymers, 13(15), 2464. under Creative Commons license.

vesicle membranes, impermeable to slightly permeable (Fig. 9.6). Such a system has shown a sustained release upon irradiation with the shorter wavelength ultraviolet (UV) radiation (Wei et al., 2016). The usage of photosensitive polymers has been explored to aid the wound healing process, which can also protect against on-site infection. In this regard, methacrylate hyaluronan polyacrylamide (MHA PAAm) hydrogels, integrated with silver nanoparticles, have been explored by a research group (Tang et al., 2020). Hyaluronic acid has a photo-crosslinking feature and is abundantly present in human tissues. The formed hydrogel attained an antimicrobial and hemostatic



CHAPTER 9 Smart polymers for biomedical applications

FIGURE 9.6 Photochromic polymersomes displaying photo-switchable and reversible bilayer permeability. Reproduced from Wei, M., Gao, Y., Li, X., & Serpe, M. J. (2016). Stimuli-responsive polymers and their applications. Polymer Chemistry, 8(1), 127 143. under Creative Commons license.

activity through a two-step process. Under UV irradiation, a free radical polymerization starts over both MHA and PAAm which forms hydrogel upon photocrosslinking. In the later stage, biodegradable gelatin (holding an amine group) was coupled with carboxylic group of MHA and finally synthesizing skin adhesive with antibacterial hemostatic activities (AHAs) (Tang et al., 2020). AHAs boosted tissue formation at the wounded site, improved vascularization and collagen formation. The in vivo study displayed a faster wound closure in the infected rat models. Another successful application of these polymers was seen in cancer chemotherapy. A spiropyran and polyethylene glycol (PEG)-based hybrid nanoparticle was loaded with docetaxel and was evaluated for tissue penetration and drug release (Tong, Chiang, & Kohane, 2013). The triggered UV (365 nm) light assists in a reversible change in the nanoparticle volume and thus influences the release. The light irradiation converts the spiropyran to merocyanine (zwitterion). The merocyanine is less stable than spiropyran and, therefore, under the influence of visible light or simply in darkness, the system reverts, resulting in an increased size of nanoparticles (Tong et al., 2013). This photo-switching assists in repeated dosing (Table 9.3).

9.6 Enzyme-responsive polymers

Table 9.3 Photoresponsive polymers in the biomedical fields. Polymer

Light irradiation effect

Poly[S-(o-nitrobenzyl)L-cysteine-ethylene glycol] (PNBC-PEO)


Collagen hydrolysate gelatin and methacrylate




Polyethyleneglycol (PEG)-star-2-(4-nitro-3benzyl carbonate camptothecin) phenoxyethyl methacrylate




Release of anticancer drug doxorubicin in a controlled manner with a change in light irradiation time The polymeric hydrogel was employed for transplantation of nucleus pulposus cells to support regeneration Higher cell viability and the possibility of the osteogenic differentiation Controlled loading of camptothecin. Possible traceable intracellular release and distribution

Liu and Dong (2012)

Silva-Correia et al. (2013)

Fedorovich et al. (2009)

Li et al. (2018)

9.6 Enzyme-responsive polymers Enzymes are a fairly new class of stimuli that polymer scientists have explored. They are crucial in various biological processes and metabolic pathways. Enzymes control a large number of dynamic processes occurring inside the human body. The idea of incorporating enzyme responsiveness to the biomaterial brings the artificial system close to the biological system. The enzyme-responsive polymers (ERPs) can alter functional changes in the polymer by responding to the small biomolecules. This widely accepted definition of ERP includes polymers whose structure and function change upon direct actions of selected enzymes (Zelzer, 2014). Various ERPs can perform innumerable tasks efficiently in their biological niche. However, this can even be a disadvantage of these polymers as the enzyme’s activity will be specific to a particular microenvironment. ERPs tend to undergo macroscopic changes through selective enzyme catalysis (Asha, Srinivas, Hao, & Narain, 2019). These catalysis reactions are substrate-specific. A few of the most significant advantages of ERP include no requirement of the trigger from the external environment, higher selectivity, and the ability to work under mild conditions (Cabane, Zhang, Langowska, Palivan, & Meier, 2012). ERPs can exist in the form of solutions, gel, self-assembled structures, nanoparticles, films, etc. (Asha et al., 2019). The design of ERP can occur either through



CHAPTER 9 Smart polymers for biomedical applications

the use of enzyme degradable polymer or simply by amending the polymeric chain with enzyme responsive, functional groups. Typically an ERP includes two subunits, that is an enzyme recognized specific substrate and another component that controls the reversible/irreversible transitions (Asha et al., 2019). ERPs need to meet three general requirements for efficient functioning. Firstly, the polymers need to have an incorporated substrate or moiety specific to an enzyme for recognition (Chandrawati, 2016). For most proteolytic enzymes, the recognition moiety is peptide or linker chains that are conjugated with one particular amino-acid sequence. Next, the substrate accessibility to the enzyme should be considered. The substrate accessibility is responsible for guiding the kinetics of the enzymecatalyzed reaction. And last is the translation of enzyme-induced response to the modification in the material’s properties like degradation or morphological alterations (Chandrawati, 2016). The intrinsic biocompatibility of enzymes allows the exploitation of ERPs for drug delivery, tissue regeneration, diagnostics, and selfhealing materials. The therapeutic agents or bioactive molecules can be incorporated through the covalent attachment or physical encapsulations for the potential application of ERP in the mentioned fields.

9.6.1 Applications Many natural and synthetic polymers are modified for enzyme responsiveness, specifically for the application of drug delivery. Hydrolase enzyme has a vital role in the hydrolysis of many biomolecules (protein, lipids, nucleic acids, etc.). Proteases, esterases, and glycosylases are three significant subclasses of hydrolase enzymes. Many diseases like Alzheimer’s, atherosclerosis, and several viral infections (e.g., influenza) are often associated with increased proteases. The design of hydrolase-sensitive polymer will function in a manner that it can specifically be cleaved by a protease and thus release the drug at the targeted site. Wilson, Salas, and Guiseppi-Elie (2012) showed a controlled and targeted delivery of pro-drug (cleavable peptide) using α-chymotrypsin (protease) inside a poly-2-hydroxyethyl methacrylate hydrogel system (Wilson et al., 2012). The peptide sequence was cleaved by the chymotrypsin, and the release of the drug occurred due to the alteration in hydrophobicity of the hydrogel. ERPs can even be employed for targeted delivery in cancer chemotherapy. In this regard, a phosphatase-responsive supramolecular spherical assembly was designed by a group of researchers that mainly utilized noncovalent interactions for its assembly. This structure was based upon the complexion of calixarene with adenosine triphosphate (Wang, Guo, Cao, & Liu, 2013). Calixarene is crucial in providing rigidity and stability to such structures and constitutes sites for enzymatic reactions (Lee, Lee, & Jiang, 2004). These spherical assemblies are quite responsive to phosphatase, which is overexpressed in many cancerous cells and thus can be employed as delivery vehicles in cancer therapy. The application of ERP is not limited to the delivery of therapeutic drugs but is also considered for peptide delivery. Antimicrobial peptides are although efficient in their activity; however, their application is often limited due

9.6 Enzyme-responsive polymers

to inactivation from serum proteases, and high cost. An enzyme-responsive polyion complex nanoparticle was synthesized for a selective delivery of antimicrobial peptide, that is PEI, which was tested in presence of infectious Pseudomonas aeruginosa (Fig. 9.7) (Insua et al., 2016). It was shown that the nanoparticle and peptide can be easily degraded by the enzyme elastase (LasB), which is secreted from the pathogen P. aeruginosa. The tissue repair and regeneration potential of ERPs are again an appreciated field. The presence of avascularity in the cartilage tissue often limits its ability of self-regeneration. The aggrecanase-degradable hydrogel was synthesized and tested with chondrocytes for application in cartilage repair. Cartilage tissue comprises elastic collagen II fibrils and proteoglycans, majorly aggrecan. The aggrecan consists of two major sites for its proteolysis termed as interglobular domain (IGD). Therefore the designed hydrogel was based on a specific cleavage site within IGD (Durigova, Nagase, Mort, & Roughley, 2011). The encapsulated cells in the hydrogel were capable of degrading the hydrogel and promoted the growth of hyaline-like cartilage, which is desired for cartilage regeneration (Skaalure, Chu, & Bryant, 2015). Matrix metalloproteinases (MMPs) are yet another class of hydrolases that are often explored for this purpose. Schneider, Chu, Randolph, and Bryant (2019) reported MMP-sensitive PEG hydrogel for their beneficial role in cartilage regeneration (Schneider et al., 2019). Since growth factors are essential for repair and regeneration, the formulated hydrogel was incorporated with transforming growth factor-β. Prepared MMP-sensitive hydrogel was able to form tissue representative of hyaline cartilage and was rich in aggrecan, decorin, biglycan, and collagen type II (Schneider et al., 2019) (Table 9.4).

FIGURE 9.7 Assembly and oxidative cross-linking of PIC nanoparticles from P1SH (Ac-C-E-GLA-E-COH) and antimicrobial branched PEI. Degradation of PIC nanoparticles by LasB and subsequent PEI release. PEI, Polyethyleneimine; PIC, polyion complex. Reproduced from Insua, I., Liamas, E., Zhang, Z., Peacock, A. F. A., Krachler, A. M., & Fernandez-Trillo, F. (2016). Enzyme-responsive polyion complex (PIC) nanoparticles for the targeted delivery of antimicrobial polymers. Polymer Chemistry, 7(15), 2684 2690. under Creative Commons license.



CHAPTER 9 Smart polymers for biomedical applications

Table 9.4 Enzyme-responsive polymers and their applications. S. No. 1.


Polymers Poly(N-[2(acryloyloxy)ethyl]-N[p-acetyloxyphenyl]N,Ndiethylammonium chloride) Naphthalene-nitric oxide (NO) hydrogel

Responsive enzyme




Intraperitoneal gene therapy and reduced adverse effects

Qiu, Gao, Liu, Wang, and Shen (2018)


Controlled release of NO improved the therapeutic efficiency of mesenchymal stem cells (MSCs). This aids in the betterment of myocardial infection Helped direct differentiation of MSCs

Yao et al. (2015)


Polyethylene glycol (PEG)



PEG and poly (styrene)



Chondroitin sulfate and PEG

Transglutaminase factor XIII

The enzymeresponsive cleavage has potential application in the colon treatment Promote tissue healing and regeneration

Anderson, Lin, Kuntzler, and Anseth (2011) Rao and Khan (2013)

Anjum et al. (2016)

MMPs, Matrix metalloproteinases.

9.7 Conclusion In recent years SPs have evolved along with great potential in the biomedical field. These are a particular class of polymers that undergo a rapid transition in their physicochemical properties based upon environmental changes. Alteration in the surrounding environment like pH, temperature, light, and magnetic or electric fields acts as a trigger for the polymeric systems. The majority of the researchers have explored various environmental triggered pH, temperature, light, and bioenzymes for the design and synthesis of SPs. The mentioned stimuli are known for their better responsiveness under in vivo conditions, biocompatibility, nontoxicity, biodegradability, and possibility of dose quantification. This chapter provided a glimpse of SPs and their utility as an intelligent biomaterial in a wide range of applications. The various examples discussed under individual topics above give a clear picture regarding the versatility of these materials. Design of


drug delivery system through SPs can improve precision delivery of bioactives and hence the efficiency of the treatment of diseased conditions. Such delivery systems have found interest in specific applications like cancer and gene therapy as well. Additionally, the role of SPs in tissue repair and regeneration is crucial, as SPs are designed to modulate hypersensitivity without hampering the immune system. SPs hold great potential and an exciting future in biomedicine, and therefore researchers keep developing and creating new SPs that are responsive to various environmental stimuli.

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CHAPTER 9 Smart polymers for biomedical applications

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CHAPTER 9 Smart polymers for biomedical applications

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Chitosan-based nanoparticles for ocular drug delivery


Kunal Pal1, Bikash K. Pradhan1, Doman Kim2 and Maciej Jarze˛bski3 1

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India 2 Department of International Agricultural Technology and Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea 3 Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznan´ ´ Poland University of Life Sciences, Poznan,

10.1 Introduction The human eyes being a protected organ, restrict the bioavailability of the drugs after the drug formulations are applied over the ocular surface (Li et al., 2018). Hence, it is challenging for pharmaceutical researchers to deliver a drug to the anteroposterior segments of the eye (Rodrigues et al., 2018; Suri, Beg, & Kohli, 2020). In this regard, several studies have explored the nanoparticle-mediated ocular drug delivery system and got efficient results (Alkholief et al., 2019; Taghe, Mirzaeei, Alany, & Nokhodchi, 2020). Various polymers, including natural polymers, have been explored to develop nanoparticle-mediated ocular drug delivery systems (Jumelle, Gholizadeh, Annabi, & Dana, 2020; Mittal & Kaur, 2019a, 2019b; Tan & Ho, 2018). Among these, chitosan is one of the most prevalent polymers that has been explored with some success (Arafa, Girgis, & ElDahan, 2020; Bao et al., 2021; De Gaetano et al., 2021). Chitosan, a cationic linear polysaccharide, has been greatly used to design formulations for nanotherapeutic applications to treat ocular diseases (Dai et al., 2020; Kazemi & Javanbakht, 2020; Yang, Cabe, Nowak, & Langert, 2022). The success of the chitosan formulations as ocular delivery systems has been related to the mucoadhesive property of the chitosan (Dubashynskaya et al., 2020; Irimia, Dinu-Pıˆrvu, et al., 2018; Sun et al., 2022). The mucoadhesive properties of chitosan could be related to the presence of the amino groups (Coutinho, Lima, Afonso, & Reis, 2020; Laffleur & Ro¨ttges, 2019; Pauluk, Padilha, Khalil, & Mainardes, 2019). These amino groups interact with the sialic acid residues that are in abundance in mucosal linings in the human body (Hejjaji, Smith, & Morris, 2018; Kolawole, Lau, & Khutoryanskiy, 2019; Pauluk et al., 2019). It has been found that the ocular retention of the chitosan-based nanoparticles can be tailored by crosslinking Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

(Abruzzo et al., 2021; Samprasit, Opanasopit, & Chamsai, 2021). The ocular retention, in turn, improves the penetration of the chitosan nanoformulations within the eye, thereby significantly improving the bioavailability of the drugs (Irimia, Ghica, et al., 2018; Li et al., 2018). Further, chitosan is biodegradable and biocompatible (nontoxic) (Islam, Dmour, & Taha, 2019; Mart˘au, Mihai, & Vodnar, 2019; Zhang et al., 2021). Accordingly, the human body degrades the chitosan nanoparticles without forming any toxic by-products penetrating the ocular tissue. In this chapter, the anatomy of the eye and types of ocular diseases will be discussed initially. Thereafter the properties of chitosan and some potential applications of chitosan nanoparticle based formulations will be briefly discussed.

10.2 Anatomy and protection mechanism of eye The human eye is nearly a spherical organ located in the orbits (Foletti et al., 2019; Glarin et al., 2021). Fig. 10.1 summarizes the different parts of the eye.

FIGURE 10.1 Schematic diagram of the anatomy of the eye and the diseases associated with the eyes. Reproduced from Vichare, R., Garner, I., Paulson, R. J., Tzekov, R., Sahiner, N., Panguluri, S. K., . . . Biswal, M. R. (2020). Biofabrication of chitosan-based nanomedicines and its potential use for translational ophthalmic applications. Applied Sciences, 10(12), 4189. under creative commons license.

10.2 Anatomy and protection mechanism of eye

Human eyes consist of anterior and posterior segments (El Basha, Furuta, Iyer, & Bolch, 2018). A layer of transparent tissue called the cornea is present in the outermost part of the anterior segment of the eye. It is in continuation with the sclera, an opaque white tissue (Lee, Low, Kim, & Teoh, 2022). The sclera helps to maintain the shape of the eye and protects it from the outer environment. On the other hand, the cornea allows visible light to enter the inner part of the eye. The eye’s anterior chamber is the front part that lies between the cornea and lens (Erdem et al., 2021). Iris forms an aperture known as the pupil, which helps to accommodate the eye in bright and dark regions by controlling the aperture opening. The iris is followed by the anterior portion of the lens, which is connected with the ciliary body. The ciliary body helps to control the curvature of the lens so as to focus the light onto the macula of the retinal tissue (Geiger et al., 2020; Yui, Kunikata, Aizawa, & Nakazawa, 2019). The posterior part of the lens forms the interface with the vitreous humor of the posterior chamber. In other words, the anterior and posterior chambers of the eye are segregated by the lens (Jacobson & Bohnsack, 2021; Nagae, Sawamura, & Aihara, 2020). The retinal tissue is present at the rear end of the posterior chamber (Peynshaert, Devoldere, De Smedt, & Remaut, 2018). The macula is the part of the retinal tissue where the image is formed (Bagewadi, Parameswaran, Subramanian, Sethuraman, & Subramanian, 2021; Inana et al., 2018). The retinal tissue forms the optic nerve, which collects information from the retinal tissue and transfers the same to the central nervous system (Mesentier-Louro et al., 2021). The eyes are prone to various ocular diseases. According to the location of the disease, the ocular diseases are categorized into the anterior and posterior segment diseases. Some of the diseases of the anterior segments include cataracts, corneal ulcers, keratitis, and conjunctivitis (Hoshi, Todokoro, & Sasaki, 2020; Stamate, T˘ataru, & Zemba, 2021). Among these diseases, cataract is one of the most commonly occurring ocular diseases across the globe. The cataract is characterized by the clouding of the lens, which is usually transparent. If left untreated, it will induce blindness in the patients and has been reported to be the leading cause of blindness (Vichare et al., 2020). Some posterior segment diseases include retinal detachment, glaucoma, macular degeneration, and endophthalmitis (Peng, Kung, Tsai, & Wu, 2021). Usually, ocular diseases are treated with topical administration of pharmaceutical formulations. However, in several cases, surgical procedures are performed. Unfortunately, there are several limitations for delivering the drugs through topical administration. The leading cause is the poor bioavailability of the drug at the site of the disease (Lynch et al., 2019). This is due to the loss of the drug formulation due to the formation of several barrier mechanisms (Fig. 10.2). Some of the barriers to the drug delivery to the eyes that hampers the bioavailability include tear film barrier (consist of an outer hydrophobic layer followed by an aqueous layer), tight epithelial junction of the cornea, reflex blinking, metabolism in ocular tissue, nasolacrimal drainage, efflux pumps, blood retinal barrier, and blood aqueous barrier (Bachu, Chowdhury, Al-Saedi, Karla, & Boddu, 2018; Bı´ro´ & Aigner, 2019). Hence, there is poor uptake of the drug molecules.



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

FIGURE 10.2 Anatomy of the eye highlighting the different ocular barriers. BAB, Blood aqueous barrier; BRB, blood retinal barrier. Reproduced from Bı´ro´, T., & Aigner, Z. (2019). Current approaches to use cyclodextrins and mucoadhesive polymers in ocular drug delivery—A mini-review. Scientia Pharmaceutica, 87(3), 15. 2218-0532/87/3/15 under creative commons license.

10.3 Properties of chitosan Chitosan is a polycationic polysaccharide. The backbone of the polysaccharide is linear. Chitosan is obtained from crustaceans (e.g., lobster, shrimp, and crab), insects (e.g., butterfly, and fly), and fungi (e.g., Aspergillus niger and Penicillium chrysogenum) (Fig. 10.3A) (Bastiaens, Soetemans, D’Hondt, & Elst, 2019; da Silva Alves, Healy, Pinto, Cadaval, & Breslin, 2021). The chitosan is synthesized from another natural linear polysaccharide, chitin. Chitin is converted to chitosan by the process of partial deacetylation (Harmsen, Tuveng, Antonsen, Eijsink, & Sørlie, 2019). The process of deacetylation can be carried out either by the chitin deacetylase enzyme or the alkaline deacetylation process (Fig. 10.3B) (Bastiaens et al., 2019). Chemically, the chitosan polysaccharide is composed of β-(1 4)-linked D-glucosamine and Nacetyl-D-glucosamine monomeric units (Resmi & Beena, 2021; Sahira Nsayef Muslim, 2018). The distribution of these monomeric units is random. The existence of a free amine group in N-acetyl-D-glucosamine is the reason behind the cationic nature of the chitosan. Chitosan can form electrostatic complexes with negatively charged polymers and other chemical entities (e.g., sodium tripolyphosphate) (Hosseini, Soleimani, & Nikkhah, 2018) due to its cationic nature. Inherently chitosan is nontoxic to human tissues. Further, it is biodegradable and biocompatible. Also, the polysaccharide exhibits antimicrobial and antioxidant properties (Fig. 10.4;

10.3 Properties of chitosan

FIGURE 10.3 (A) Sources of chitosan and (B) conversion processes to synthesize chitosan from chitin. Reproduced from da Silva Alves, D. C., Healy, B., Pinto, L. A. D. A., Cadaval, T. R. S. A., & Breslin, C. B. (2021). Recent developments in chitosan-based adsorbents for the removal of pollutants from aqueous environments. Molecules, 26(3), 594. under creative commons license.

FIGURE 10.4 Inherent properties of chitosan that makes it suitable for biomedical applications. ˇ & Leitgeb, M. (2019). Chitosan-based (nano)materials ˇ c, ˇ M., Knez, Z., Reproduced from Kravanja, G., Primozi for novel biomedical applications. Molecules, 24(10), 1960. under creative commons license.

Kravanja, Primoˇziˇc, Knez, & Leitgeb, 2019). These inherent properties of chitosan make it an excellent material for biomedical and pharmaceutical applications (Ali & Ahmed, 2018).



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

Though chitosan exhibits several advantageous properties, some of the properties are more beneficial in ocular delivery than others. The most important properties of chitosan that play an essential role in ocular delivery include mucoadhesion, antibacterial, and penetration enhancement (Zamboulis et al., 2020). The mucoadhesive property of chitosan is exhibited due to the presence of hydroxyl and amine functional groups. These functional groups can form noncovalent bonds (ionic and hydrogen-bonding interactions) with the mucin, which is the primary constituent of the mucosal layers. Again, these noncovalent interactions improve the adhesion of chitosan formulations over the mucosal layers, including the ocular surface. Mucin is a negatively charged polymer, which is ascribed to the presence of free carbohydrate-bound ester sulfate residues and the carboxyl groups of sialic acid groups (Zamboulis et al., 2020). The interactions of chitosan nanoparticles with the mucosal layers and subsequent drug delivery mechanism is summarized in Fig. 10.5 (Mohammed, Syeda, Wasan, & Wasan, 2017). As mentioned in the previous paragraph, there is an ionic interaction between the mucosal layer and the chitosan nanoparticles. This interaction significantly

FIGURE 10.5 Schematic showing the interaction of chitosan nanoparticles with the mucosal layer and subsequent drug delivery. Reproduced from Mohammed, M. A., Syeda, J. T. M., Wasan, K. M., & Wasan, E. K. (2017). An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics, 9(4), 53. https:// under creative commons license.

10.3 Properties of chitosan

improves the bioadhesivity of the chitosan nanoparticles over the mucosal surface, thereby improving their ocular residence time by preventing lacrimal elimination. The improved residence time of the chitosan nanoparticles over the ocular surface increases their chances of transcellular transport within the ocular tissues. Transcellular transport is made possible due to the disruption of the tight junctions (Hong, Yoo, Kim, & Lee, 2017). The schematic representation of the transcellular transport of the chitosan nanoparticles is depicted in Fig. 10.6. Chitosan can elicit antimicrobial activity in various ways (Ke, Deng, Chuang, & Lin, 2021). The high molecular weight chitosan can chelate with the ions and nutrients around the microbes. Accordingly, the microbes will be deprived of the required ions and nutrients to survive and grow. Further, such chitosan molecules can interact with the lipoteichoic acids present in the peptidoglycan layer of Gram-positive bacteria, thereby weakening their activity. Also, the chitosan with higher molecular weight can disrupt the cell membranes of Gram-positive bacteria, Gram-negative bacteria, and fungi (Ke et al., 2021). It causes the leakage of the internal contents of the microbes, thereby resulting in their death. On the other hand, the chitosan molecules with a low molecular weight influence penetrate the cell wall of the microbes. After their penetration, they hinder the functions of DNA/RNA. The protein synthesis within the microbes can also be significantly affected. In fungi, low molecular weight chitosan has been reported to inhibit

FIGURE 10.6 Schematic representation of the transcellular transport exhibited by chitosan nanoparticles. Reproduced from Hong, S.-C., Yoo, S.-Y., Kim, H., & Lee, J. (2017). Chitosan-based multifunctional platforms for local delivery of therapeutics. Marine Drugs, 15(3), 60. 3/60 under creative commons license.



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

FIGURE 10.7 Schematic representation exhibiting the antimicrobial activity of chitosan molecules against: (A) Gram positive bacteria, (B) Gram negative bacteria, and (C) fungi. Reproduced from Ke, C.-L., Deng, F.-S., Chuang, C.-Y., & Lin, C.-H. (2021). Antimicrobial actions and applications of chitosan. Polymers, 13(6), 904. under creative commons license.

mitochondrial activity (Ke et al., 2021). Fig. 10.7 depicts the mechanism involved with the antimicrobial activities of chitosan.

10.4 Some recent applications of chitosan nanoparticles in ocular delivery Clarithromycin-loaded chitosan nanoparticles have been prepared by (Bin-Jumah et al., 2020). The drug clarithromycin is a well-established antibiotic and is characterized as a broad-spectrum macrolide antibiotic. It has been reported that the antibiotic is widely used to treat various bacterial keratitis infections.

10.4 Some recent applications of chitosan nanoparticles

The nanoparticulate formulations showed sustained drug release properties. Further, the corneal permeation of the drug was considerably increased as compared to the clarithromycin solution. An improvement in the corneal permeation was reasoned to the increased precorneal residence time. Even though the corneal permeation of the drug was enhanced, the formulations did not cause damage to the corneal tissues. The developed formulation was proposed to treat bacterial conjunctivitis. In Shinde, Joshi, Jain, and Singh (2019), authors have reported the synthesis of N-trimethyl chitosan nanoparticles for the ocular delivery of flurbiprofen, a nonsteroidal antiinflammatory drug. The solubility of flurbiprofen was first improved by developing a complex with hydroxyl propyl-β-cyclodextrin. Thereafter the complex was loaded into the chitosan nanoparticle matrices. These nanoparticulate systems were capable of transmucosal delivery of flurbiprofen over a prolonged period of time. Ameeduzzafar, Imam, Abbas Bukhari, Ahmad, and Ali (2018) have developed a levofloxacin-loaded chitosan nanoparticle for ocular drug delivery. Levofloxacin is a well-established antibacterial agent that has been successfully explored to treat ocular infections. The nanoparticles were prepared by the ionotropic gelation method. Herein, the authors used sodium tripolyphosphate as the ionic crosslinker. The optimized nanoparticle formulation was converted into an in situ gel-forming formulation. As the encapsulation efficiency of the drug within the nanoparticles was very high, it improved the loading efficiency of the drug. It was found that the corneal residence of the chitosan nanoparticles was enhanced when converted into an in situ gel formulation. Further, the chitosan nanoparticles did not induce irritation of the ocular tissue. The proposed formulation showed sufficiently high antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Also, the proposed formulation achieved a shelf life of 2.16 years, which is high enough to promote its commercialization. In a similar study, gentamycin-ferrying chitosan nanoparticles were used to develop in situ gel formulations (Alruwaili et al., 2020). The in situ gel was pH-sensitive. It was prepared using the polymer carbopol 974P, a well-known pH-sensitive polymer. This polymer has been extensively used to develop several pharmaceutical products, including ocular delivery systems. In a recent study, Shahab, Rizwanullah, Alshehri, and Imam (2020) have developed chitosan-decorated polycaprolactone nanoparticles (Shahab et al., 2020). The developed nanoparticles, prepared by single-step emulsification technique, were explored to improve ocular drug delivery of dorzolamide. Dorzolamide is used to reduce the ocular pressure in open angle-type glaucoma. The drug has also been used to treat ocular hypertension. The release of the drug from the nanoparticles followed a biphasic release pattern. During the first 2 h, a burst release of the drug was observed. Thereafter a sustained release of the drug for 12 h was revealed. The results showed an improved corneal permeation of the drug. It can be explained by the higher mucoadhesive properties of the developed nanoparticulate formulations. Finally, the nanoparticles were found to be safe for ocular administration.



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

Table 10.1 Recent publications on the applications of chitosan-based nanoparticle formulations for ocular drug delivery.


Composition of the formulation

Nanoparticle preparation method

Ocular disease focused

Zhao et al. (2017)

Timolol maleate loaded galactosylated chitosan

Ionic crosslinking


Silva et al. (2017)

Chitosanhyaluronic acidbased nanoparticles containing ceftazidime

Ionotropic gelation

Bacterial keratitis

Wang et al. (2018)

Chitosan-coated solid lipid nanoparticles encapsulated with methazolamide

Emulsificationsolvent evaporation method


Imam, Bukhari, Ahmad, and Ali (2018)

Levofloxacinloaded chitosan nanoparticle

Ionic gelation

Ocular infection

Bin-Jumah et al. (2020)

Clarithromycinloaded chitosan nanoparticles

Iontophoretic gelation

Ocular infection

Inference from the study The proposed formulation showed a sustained release effect and enhanced penetration and retention in the cornea compared to the commercially available eye drop. The nanoparticle formulation presented showed the necessary mucoadhesive property desirable for efficient drug delivery. The nanoformulation also possesses good antimicrobial properties. The proposed formulation showed an efficient result in the ocular delivery of methazolamide. A significant decrease in the intra-ocular pressure was also observed. The optimized formulation shows nonirritability for the cornea. Also, the formulation is found to possess a higher antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. The optimized formulation showed a small particle size, high encapsulation, and a sustained drug release profile. (Continued)

10.5 Conclusion

Table 10.1 Recent publications on the applications of chitosan-based nanoparticle formulations for ocular drug delivery. Continued Nanoparticle preparation method

Ocular disease focused


Composition of the formulation

Sobhani, MohammadiSamani, and Arazi (2020)

Chitosansulfacetamide sodium nanoparticles

Iontophoretic gelation

Ocular infection

Yu et al. (2020)

Dexamethasoneglycol chitosan nanoparticles

Iontophoretic gelation

Ocular diseases

Inference from the study The proposed formulation showed that sulfacetamide sodium did not lead satisfactory drug release profile during the 24 h test. The proposed nanoparticle formulation showed good ocular tolerance and longer precorneal duration than the aqueous solution.

In an interesting study, lipid nanoparticles were coated with chitosan (Eid, Elkomy, El Menshawe, & Salem, 2019). The coated nanoparticulate systems were explored for the ocular delivery of ofloxacin. It was observed that 66% of the drug permeated through the cornea. The authors reported that chitosan coating significantly improved trans-corneal permeation and bioavailability. It is due to an increased mucoadhesive property. Table 10.1 lists some of the recent publications based on chitosan-based nanoparticle formulations.

10.5 Conclusion Chitosan is a naturally occurring polymer and is obtained from chitin by the process of deacetylation. This polymer is a versatile polymer and has been explored in various applications, including food, pharmaceutical, and biomedical. The main reason behind the versatility of this polymer is its inherent biocompatible, biodegradable, and mucoadhesive properties. Also, chitosan is nonirritant to the human cells and tissues and does not provoke an immunological response. Further, the polymer has been found to enhance the transmucosal penetration of the drug molecules. Due to these properties, it has found applications in ocular drug delivery. Chitosan and its derivatives can considerably improve the ocular residence time, thereby improving the bioavailability of the drugs at the ocular tissue. Accordingly, there is an increased therapeutic effect of the drugs. The effect is more pronounced when the polymer is used to develop nanoparticles. This therapeutic



CHAPTER 10 Chitosan-based nanoparticles for ocular drug delivery

effect of chitosan nanoparticles is due to their small size. Also, the surface area of such nanoparticulate systems is quite high, which in turn increases the mucoadhesive property of the polymers. Even though various synthesis methods of chitosan nanoparticles are present, the ionotropic gelation method is the most common method. Among the various ionic crosslinker, sodium tripolyphosphate has quite often been employed. This is because the development of chitosan nanoparticles using sodium tripolyphosphate is very convenient. Such nanoparticles have found applications as sustained release systems for ocular drug delivery. The ocular residence of these nanoparticle formulations can be further increased by converting them into in situ gelforming systems. Researchers have reported many encouraging results based on chitosan coating over other polymeric nanoparticulate matrices. In gist, it has been found that chitosan and its derivatives can be explored to develop novel ocular delivery systems with improved therapeutic efficacy.

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Appraisal of conducting polymers for potential bioelectronics


Rimita Dey1 and Pallab Datta2 1

Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology, Howrah, West Bengal, India 2 Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India

11.1 Introduction Discoveries in polymer chemistry showing that polymers can conduct electricity open new path for application of polymeric materials in electronics industries. Before the 1970s all carbon-based polymers were considered electrical insulators. Thereafter electronic polymers are discovered which facilitate in energy conversion and storing in various systems like rechargeable batteries, solar cells, and ultracapacitors. Electronic polymers are highly appreciated because of optical properties, high electrical conductivity, mechanical flexibility, and affordable cost (Granero, Wagner, Wagner, Razal, & Wallace, 2011; Weng, Pan, Wu, & Chen, 2015). Intrinsically conductive polymers (ICPs) are obtained by a two-stage process—(1) the monomer is transformed into a conjugated double-bonded polymer; and (2) the extra positive or negative charge is added by a doping method. To conduct the entire process in single step, excess charge is introduced during polymerization process. Conjugated polymers are doped via photochemical, chemical or electromechanical route. Charge transfer between dopant and polymer is the characteristics of doping reactions. The most common method of chemical synthesis is step growth condensation polymerization to produce polyaniline, polyparaphenylene, and polythiophene (and its derivatives). Polyacetylene is generally synthesized by polymerizing acetylene using ZieglerNatta method. Polymers (like polyaniline, polypyrrole, and polythiophene) are prepared by electrochemical oxidation methods where the monomer is anodically polymerized on electrode surface using appropriate solvent or electrode medium. The chemical structures of the repeating units of conducting polymers can exhibit much variation (Min, Patel, & Koh, 2018) and some of them are shown in Fig. 11.1. Polymer film is synthesized by the oxidation reaction. Undoping of the film can be done by changing the current flow. The resultant doped polymers can be easily stripped off from the surface of the electrode in coated and self-supporting film form and Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 11 Appraisal of conducting polymers

FIGURE 11.1 Chemical structures of repeating units of some common conducting polymers. Reproduced from Min, J.H., Patel, M., & Koh, W.-G. (2018). Incorporation of conductive materials into hydrogels for tissue engineering applications. Polymers, 10.

thus electrochemical polymerization method is more preferable to chemical methods. Photochemical synthesis techniques for synthesis of ICPs are not preferable but they are applied for polymerizing pyrrole and some derivatives of thiophene. Thin polymer films can be fabricated from dedicated solid surfaces such as conductive indium tin oxideplated glass. Distinctive coexistence of electrical and mechanical properties can be found in conjugated polymers. Most conjugated polymers are insoluble and infusible in nature due to their chemical structure. But those materials are preferred that are fusible and soluble in common solvents, enhancing easy transformation of the conductive polymers into convenient products. The polymers can be synthesized with different solid-state structures and according to the structures and properties of different polymers associated with electronic devices, the charge transport property of amorphous material occurs by several hopping events. The conformational defects of each chain determine the structure of polymers. For example, due to very slow equilibration, the definite shape of less molecule polymers like poly(p-phenylene vinylene) (PPV) becomes highly organized, and this does not affect the association between conformation and electronic configuration (Qin and Troisi, 2013). In the crystalline regions of the electronic polymers, the chains are folded and orderly arranged. The flow of charge through electronic polymer is hindered by interspherulite boundaries because of reduction in tie chains which connect adjoining structured regions in comparison with edges between quasiparallel lamella inside the spherulite (Street, Northrup, & Salleo, 2005). Moreover, fabrication of different morphologies like nanofiber, nanoparticles, etc. can further provide better control on the stability and morphology. Other than core shell, hollow nanoparticles and solid structure are becoming famous in recent years. Polymer-coated hollow sulfur particles (transfers the electron and prevents the spill of polysulfides) are manufactured by in situ polymerization and used in lithiumsulfur batteries as cathode materials (Li et al., 2013).

11.2 Sensors and actuators used on conducting polymers

Polyaniline (PANI) is commonly used material due to its simplistic synthetic process, tailor ability by nonredox acidic or basic doping, more environmental stability, ease in controlling electrical conductance, and probability of a large-scale fabrication and is highly studied for electrorheological, electronic, and electrochemical applications. Monodispersed PANI nanoparticles are obtained from oxidative dispersed polymerization where poly(sodium 4-styrenesulfonate) (PSSA) is employed as a dopant agent as well as polymeric stabilizer due to its activity. Apart from nanoparticles, nanofibers have been immensely explored for structural characteristics such as high curvatures and large aspect ratios that offer higher distinct surface areas and removable electrochemical activities. Nanofiber networks or arrays are used in biological materials, composite materials, and filtering membranes other than a variety of electronic materials. Regio-regular P3HT is one of the important organic semiconductors widely used in fabricating electronic devices like solar cells, field-effect transistors, or different sensors. P3HT-based nanofibers are fabricated by electrospinning and self-assembly process. Continuous and long nanofibers produced by electrospinning process are of diameters ranged from 10 nm to submicron. During fabrication of nanofibers, conducting P3HT is mixed with nonconducting polymer which decreases the electrical conductivity of P3HT. ICPs can also be converted into films having both high toughness and strength. These conducting polymers are lightweight and flexible than the other equivalent inorganic materials. Porous conducting polymers have also been explored due to high specific surface area. Pure poly(3,4-ethylenedioxythiophene) (PEDOT):PSS shows conductivity of 0.24 S/cm which is very low, but addition of nonionic surfactant stimulates the growth of PEDOT nanofibrils during coatings, which increases the conductivity (100 S/cm). After the synthesis of polymers and their fabrication in suitable form, the materials are characterized thoroughly for wide range of properties. For example, mechanical properties of the electronic devices determine their stability. For determining electronic properties of the conducting polymer, the space chargelimited conduction is considered, which means under applied voltage, the electric field from adjacent carriers dominates. Polymer-based electronic devices have high electrical conductivity. But still they are greatly sensitive to few reaction parameters, for example, temperature, impurities, and moisture content which sometimes form heterogeneous surface properties and severely hamper the electrical conductivity and reproducibility. Hence, the electronic polymers are needed to become more stable and more durable in different conditions.

11.2 Sensors and actuators used on conducting polymers Electrochemical actuator converts electrical energy into mechanical energy through electrochemical mechanisms. These actuators have huge potential in the



CHAPTER 11 Appraisal of conducting polymers

field of biomimetic sensors and devices, artificial muscles, robotics (Bauer et al., 2014; Ma, Chirarattananon, Fuller, & Wood, 2013; Madden, 2007). They are made up of shape memory alloys and inorganic materials like ferroelectric ceramics so get major attention because of their rapid responsiveness and powerful stress output (Madden et al., 2004; Zhang & Li, 2012). But they are not good for practical applications because of high cost, heavyweight, and small actuation strain (,10%). Electrochemical mechanical actuators are composed of composites of polymeric materials and are widely explored since they inherit advantages of its constituents, such as low cost, large strain, high flexibility, and synthesis controllability. Depending on mechanism of actuation, the polymeric materialsbased electromechanical actuators are classified into three types. The classes are electrothermally driving actuators, electrostatically driving, and electrochemically driving. In actuators that are driven electrostatically, various polymeric materials possessing large value of dielectric constant, for example, fluoroelastomers, isoprene, polyurethanes (PUs), polydimethylsiloxane (PDMS) are used as dielectric layer (Mirfakhrai, Madden, & Baughman, 2007). Under electric field of 412 kV/mm, the actuator comprising prestretched VHB-4910 elastomers from 3 M can induce maximum actuation strain of 158%. But they have a limited range of ultracharge voltage of operation upto 100 V/μm. There are two ways for reducing the operational voltage, one is directly to increase dielectric permittivity or to decrease the thickness for enhancing the electric field strength. To reduce the operational voltage, great measures are taken to increase films dielectric constant. Amalgamation of organic dipole groups, ceramic nanoparticles having high dielectric permittivity into polymer composition effectively enhances the dielectric permittivity of polymer film. For example, by solution mixing method, silicone oillaminated TiO2 nanoparticles are blended with SEBS (poly-styrene-co-ethylene-co-butylene-costyrene) (Stoyanov, Kollosche, Risse, McCarthy, & Kofod, 2011). Chemical grafting method is used to add dipole into the elastomer matrix at molecular level rather than conventional compositing or blending method (Kussmaul et al., 2011; Risse, Kussmaul, Kru¨ger, & Kofod, 2012). Homogenous elastomer films having better efficiency are fabricated using this method and the aggregation problem is also resolved. For example, by one-step film formation mechanism, N-alkyl-Nmethyl-p-nitroaniline, is inserted into silicone cross linkers (Kussmaul et al., 2011). Since after applying external voltage, the dipoles align among themselves and the resultant silicone having a dipole content of 13.4% exhibit the electric permittivity which is 5.9 times enhanced. The actuation performance depends on the dielectric films prestretching treatment (Zhao & Wang, 2014). Circularly or spherically, uniaxially, or biaxially, the prestretching is done upon the dielectric films. The direction of actuation can be regulated by altering the directions of prestretching. However, the actuators made from prestretching film have disadvantages such as necessity of rigid frame for maintaining the stretching state. This enhances the equipment mass leading to the work/power density reduction.

11.2 Sensors and actuators used on conducting polymers

In electrostatic actuators, due to the actuation performances, the electrodes have a significant importance. Ideally, the characteristics of materials used as electrodes are high mechanical stretchability and electrical conductivity along with properties such as stable and uniform charge distribution on both of the surfaces. For electrostatic actuation, in recent years, several stretchable electrode materials are examined. In earlier days, thin metal films were used since they have high electrical conductivity. But later they lost potential due to disadvantages of small strain less than 1%. In another way, elastomer surfaces coated with palladium, gold, or titanium nanoparticles through metal ion implantation method can be used as electrode. These gold nanoparticles laminated dielectric elastomers are stretchable up to strain of 175% and exhibit area resistance (Yun et al., 2012). For preparing high conductive stretchable electrodes, metallic nanowires are inserted into the elastomeric matrix. Carbonaceous materials like carbon power, graphite, and carbon grease are inexpensive, compatible with the dielectric elastomers and highly conducting compared to metal-based electronics. Graphene and carbon nanotubes (CNTs) also have a lot of potential as electrode materials. Single-walled CNT-based electrodes are flexible and transparent and thus the resultant actuator having self-clearing property can create 200% strain in the area. The aligned CNT sheet-based actuators show large actuation strain and directional motions. In the fields of mechanical machines, robotics, and artificial muscles, electrostatically driving actuators have huge potential (Anderson, Gisby, McKay, O’Brien, & Calius, 2012). But high operational voltage ranging from kilovolts to megavolts limits their application. So, extensive research is going on to create novel dielectric elastomers. Due to low voltage, the actuators which are driven electrochemically are widely explored (Kong & Chen, 2014). These actuators comprising electrolytic layer are sandwiched between two layers of electrode. Expansion of electrode layers and asymmetrical volume shrinkage due to migration of ions under electric field results in bending motion. For fabrication of electrochemically driven actuators, composites of ionic electroactive polymers are used. Conducting polymers (CPs) and ionic polymer metal composites (IPMC) are the mostly known among them. In case of PPy in neutral state (Carpi, Kornbluh, Sommer-Larsen, & Alici, 2011), reduction and oxidation take place respectively at the cathode and anode simultaneously. In case of actuators that are driven by anions, volume starts to expand as soon as the anions are introduced into the oxidized PPy at cathode for neutralizing desertion of electron. Conversely, reverse actuation occurs which is driven by cations when the anions are too heavy to migrate in electrolyte and at the cathode volume is contracted. The CP-based electrochemical actuator is fabricated with bilayer or multilayer designs for achieving bending motion electrochemically. For electrochemical actuators the largely used CPs are derivatives of polythiophene (PTh), polyaniline (PANI), and polypyrrole. The multilayered structured actuators are fabricated by coating two film conductive polymers with a layer of electrolyte (Torop, Aabloo, & Jager, 2014). Migration rate is the number of migrating ions per unit time. In electrolyte system, migration rate linearly



CHAPTER 11 Appraisal of conducting polymers

controls the actuation speed. During the electrochemical process, speed of migration of ions is slow; hence the strain rate of traditional conductive polymers is low. However, due to the conductive polymer’s structural instability during the cyclic electrochemical process, the conducting polymer-based actuators have short life span. To overcome this drawback, conductive or functional additives are added into the conductive polymer to increase the actuation performance. Unfortunately, the conductive polymer-based electrochemical actuators have disadvantages such as limited lifetime and delay in responsiveness. Especially, the conductive polymers’ mechanical performances in electrolytic medium degrade compared to the dry-state counterparts and this makes them unsuitable for practical applications (Spinks, Mottaghitalab, Bahrami-Samani, Whitten, & Wallace, 2006). Generally, actuators based on IPMC are electrochemically driven, comprising an ion exchange polymeric film coated by two electrodes. Contraction and expansion of electrodes create bending motion. Earlier, liquid electrolytes were majorly applied in the IPMC actuator’s operation system. Unfortunately, due to the complex electrochemical system and heavyweight, liquid electrolyte reduces the applications of electrochemical actuators. Additionally, due to liquid resistance and slow migration of ions, the actuation responsiveness is slow for liquid electrolyte. The solid-state electrolyte-based actuators can operate with stable actuation in air, replacing the liquid electrolytes (Lee et al., 2014). Due to limited conductivity, actuation responsiveness of the solid-state electrolyte-based actuators is comparatively low (Baughman, 2003). Recently, an electrolyte force strategy is adopted for fabrication of IPMC actuators by introducing polymeric or metallic interface (Detsi, Onck, De, & Hosson, 2013). Similarly, poly(vinyl alcohol)-based polymer electrolytes have also been developed for electrical double-layered capacitor application (Aziz et al., 2021) (see Fig. 11.2). When sulfuric acid doped PANI doped with porous gold electrodes, the resultant electrochemically driven actuator shows three times greater strain rate compared to the conventional threecomponent actuators. The layered structure of the electrochemically driving actuator is mostly investigated for generating bending actuation (Chen et al., 2011; Chen, Liu, Hu, & Fan, 2008; Zhang et al., 2014). In comparison to electrothermally driven actuation of inorganic components, the counterparts based on polymeric materials show superior properties such as large deformation, lightweight, and flexibility. The electrothermally driving polymer-based actuators are divided into two categories. First one is single-layer polymeric film comprising conducting additives. Due to electrothermal heating, these additives can contract or expand (Chen et al., 2008; Hu, Chen, Lu, Liu, & Chang, 2010; Sellinger, Wang, Tan, & Vaia, 2010). The other one is bilayer structured film with coating consisting of polymeric materials and current conducting layers (Chen et al., 2010; Liang et al., 2012; Zhang et al., 2011; Zhang et al., 2014; Seo, Kang, Kim, & Kim, 2012). When electric current is applied, the conducting layer having low value of coefficient of thermal expansion (CTE) can produce heat and the polymer layer expands. This leads to

11.2 Sensors and actuators used on conducting polymers

FIGURE 11.2 A capacitor design based on conducting polymers and their performance (Aziz et al., 2021). Reproduced from Aziz, S.B., Asnawi, A.S.F.M., Abdulwahid, R.T., Ghareeb, H.O., Alshehri, S.M., & Ahamad, T., et al. (2021). Design of potassium ion conducting PVA based polymer electrolyte with improved ion transport properties for EDLC device application. Journal of Materials Research, 13, 933946.

dissimilar changes in volume of the electrode materials and the polymeric layers generate bending deformation. The expansion layers are generally made up of PDMS, chitosan, PU, and epoxy. CNTs have electro heating characteristics, high electrical conductivity flexibility, so they are employed as heating electrode. For example, to form highly conducting networks, the CNTs are synthesized into the polymeric film, for example, elastomers of silicone and chitosan (Chen et al., 2008). Periodic heating of the CNTs’ conducting network causes thermal contraction and expansion and when a pulse voltage is applied, the resultant composite film actuator produces tunable vibration. Aligned CNT sheets and fibers have higher mechanical performances and high electrical conductivity. Hence, in electrothermally driving actuators, they have more potential for acting as heating electrodes. Graphene and its derivatives are also used as electrodes due to their improved mechanical, electronic, and thermal performances and negative CTE. Graphene-on-organic microactuator can generate large bending vibrational motion at driving voltage less than 4 V. Within 0.02 s the bending movement gets finished which is 10100 times quicker compared to the traditional bilayered electrothermally driving actuator. Graphene can be fabricated from graphite through chemical exfoliation mechanism (Liang et al., 2012). Resultant film of graphene is current conducting and flexible, thus it can be used as heating electrodes. CNT fibers are mostly employed for rotary and contractive actuations. The helically aligned CNT fibers have huge potential in fabrication of the electrothermally



CHAPTER 11 Appraisal of conducting polymers

driving actuator. The electrothermal fiber actuators prepared using inorganic nanowires and CNTs have very high cost. Therefore they are not used for applications purpose. As a replacement the inexpensive nylon, polyethylene and polyester are converted into helical structures and then they are used (Haines et al., 2014). The resulting coiled fibers precoated with conducting components when subjected to electrothermal treatment and heating are able to produce 49% contractive strain. For preparing supercapacitors the materials employed as flexible electrodes are conductive polymers having high capacitance and electrical conductivity (Wang et al., 2011). Various conductive polymers, for example, polythiophene (PTP), PANI, and polypyrrole (PPy) are inspected. But, after chargingdischarging mechanism, bare conductive polymeric materials generally become less stable. To overcome this obstacle, one procedure is incorporating conducting materials having superior thermal and mechanical stability and better electrical conductivity, for example, graphene and CNT. Properties like long cyclic stability, high electrical conductivity, and specific capacitance are exhibited by these hybrid electrodes. PANI is grown on the exterior part of CNT network which acts as template. PANI is synthesized after polymerizing aniline monomers by in situ chemical polymerization method and it forms a homogenous lamination on to the CNTs. To prepare thin, flexible, and freestanding CNT/PANI hybrid film, “skeleton/skin” procedure is followed. In case of flexible supercapacitors, without using extra polymeric substrate or metallic current collector, the CNT/PANI composite film is employed as both charge collector and electrodes (Niu et al., 2012).

11.3 Energy storage from conducting polymer Development of energy storing systems [namely, supercapacitors or lithium-ion batteries (LIBs)] is vital to utilize renewable and sustainable energy sources. Supercapacitors are able to provide greater magnitude of energy density compared to dielectric capacitors. Not only that they have better energy storage capacity and but also can dispatch ample amount of charges within a few seconds. Hence, they have greater power capacity compared to batteries. A supercapacitor comprises two electrodes immerged into electrolytic solution having appropriate divider (Jost, Dion, & Gogotsi, 2014). Several kinds of conductive polymers are used in pseudocapacitors in terms of electrode materials. These conducting polymers are fabricated either electrochemically or chemically by oxidizing suitable monomer (Li, Bai, & Shi, 2009; Yuan et al., 2013). Based on the charge storing method of conducting polymers, other than active materials, the reversible, rapid redox reactions occur at electrodeelectrolyte interface, raising attention in designing nanostructured CPs. Reduction in the size of bulk CPs minimizes the transportation distance for both electrons and ions that uphold the high capacitance at high density of current

11.3 Energy storage from conducting polymer

(Zhang, Uchaker, Candelaria, & Cao, 2013). Perpendicularly ordered PANI nanowire arrays show higher capacitance (950 F/g) and can preserve upto 780 F/g even at higher current densities (Wang, Wu, Meng, & Wei, 2014). This indicates that fabrication of one-dimensionally aligned nanostructures, especially nanowires, constitutes an efficacious procedure for highly performing electrodes in supercapacitors. The reason for this is accredited to the one-dimensional nanostructured PPy that permits an effective transportation of charge and delivers a minimized ion transport distance. But, there are few demerits for applying conducting polymers as materials of electrode in supercapacitors as the electrodes generally have comparatively poor cycle stability (which is responsible for inferior mechanical stability) and low electrical conductivity throughout long chargedischarge mechanisms. Although to design different composites of conducting polymers combined with other metals, metal and metal oxides/hydroxides and carbon materials overcome the drawback. Frequently, metals are employed as matrices for CPs because of their high electrical conductivities. The resulting composite electrodes generally have great performance rate, good cycling stability, and higher energy density compared to the pure CPs (Chen et al., 2015; Huang et al., 2015; Zhang, Hu, Yao, & Ye, 2015). Amidst them, stainless steel, aluminum (Al) foil, copper (Cu), nickel (Ni) foam, and gold (Au) are mostly applied metals. For example, core composite of PPy shell/3D-Ni can be fabricated using three-dimensional nickel films as current collector (Chen et al., 2015). The electrode composite exhibits high specific capacitance (726 F/g) and superior rate at high current density compared to pure PPy. Three-dimensional metal substrates with high conductivity deliver shorter diffusion route to transport ions fast increasing efficiency of transfer of electrons. Low conductivity of pure conducting polymers has moderately limited their applications in electrode materials in practical field. Conversely, carbon materials have advantages such as higher mechanical and electrical characteristics, high stability, and cost-effective (Wang et al., 2014; Yan, Wang, Wei, & Fan, 2014). Hence, blending of conducting materials and carbon materials at the molecular levels enhances the electrical conductivity and mechanical characteristics of polymers. Porous carbon materials in several forms (Chmiola, Celine Largeot, Simon, & Gogotsi, 2010; Kajdos, Kvit, Jones, Jagiello, & Yushin, 2010; Ma, Liu, & Yuan, 2013; Pech et al., 2010; Zhang & Zhao, 2009; Zhu et al., 2011). For example, mesoporous carbon, carbon sphere, activated carbon, and carbon onion constitute advantage of having high porosity, conductivity, and surface area that make them potential candidates for fabricating electrode materials. The effects of significant collaboration between porous carbon materials and CPs result in better electrochemical characteristics higher than the independent components (Liu et al., 2014; Yan et al., 2013; Nyholm, Nystro¨m, Mihranyan, & Strømme, 2011; Wang, Tao, An, Wu, & Meng, 2013; Yan, Cheng, Wang, & Li, 2011; Zhang, Kong, Cai, Luo, & Kang, 2010). By chemical oxidation polymerization method, perpendicularly aligned PANI nanowhiskers are fabricated on the superficial layer of aligned mesoporous carbon (CML-3) and the resultant PANI/CMK-3 complex



CHAPTER 11 Appraisal of conducting polymers

exhibits specific capacitance and high capacitance detention even after 1000 cycles. These improvements are electrochemical properties due to the ordered pseudocapacitance of PANI nanowhiskers which can create electrolytic ions more electrochemically accessible and decrease their transportation distance. 1D nanostructures (e.g., nanorods, nanotubes, nanowires) are coming up as new material used in supercapacitors as they impart an efficient route for transporting charges as well as minimized ionic and electronic transportation distances (Yan et al., 2014; Yu, Tetard, Zhai, & Thomas, 2015). CNTs exhibit higher aspect ratio along with higher stability and electrical conductivity compared to porous carbon material. They have open tubular network structure and superior mechanical flexibility, which helps them to become better substrate to deposit active materials. CPs in several configurations of nanosheets, nanowire/rod, and nanoparticles are homogeneously sprouted on CNT surfaces by electrostatic and ππ interactions. Other than CNTs, carbon nanofibers are easily available commercially, precisely chosen as the current collector because they have properties, for example, lightweight, flexibility, chemical stability, and superior electrical conductivity (Chen et al., 2011). Carbon cloth synthesized from nanofibers is also used for developing electrodes. It is observed that carbon cloth containing PANI nanowire dispatches extraordinarily higher specific area and gravimetric capacitance (Horng et al., 2010). The composite electrodes with magnificent electrochemical properties are more preferred as supercapacitors. Graphene as attracting 2D carbon compound shows various intensity characteristics, for example, high flexibility, high surface area, and conductivity. Hence, graphene is extensively used as a superior supporting framework to grow different CPs (Wang et al., 2014; Yan et al., 2011; Liu, Ma, Guang, Xu, & Su, 2014; Mini, Balakrishnan, Nair, & Subramanian, 2011; Xu, Sun, & Gao, 2011). For further improving electrochemical properties of electrode composites, significant labor is given for controlling the surface structure of CPs grown on nanosheets of graphene. Both graphene and CNT show higher electrical conductivity and surface areas, so their assemblage in a 3D porous structure, for example, sponge, carbon foam, framework, and aerogel incorporates their characteristics and makes highly performing supercapacitor. Three-dimensional porous nanostructures having distinct meso/micropores and high surface area make adequate contact between active materials with electrolyte and minimize the ion transportation length, which results in faster reaction kinetics, essential for superior power density. Highly ionic and current conducting active CPs decrease polarization rate and enhance the reaction kinetics, when electrical conductivity is taken into consideration. The synthesis of tertiary composites incorporates the properties of individual constituents (CP, metal oxide hydroxide, and carbon material) such that every constituent contributes their best properties in the composite (Xia et al., 2012; Grover, Shekhar, Sharma, & Singh, 2014; Tang, Han, & Zhang, 2014; Wang, Yang, Huang, & Kang, 2012; Jung, Yoon, Kim, & Rhee, 2005). It is observed that the quality of graphene/MnO2 textile electrodes is boosted up when it is wrapped with conducting PEDOT:PSS thin layer by simple dry dipping method

11.3 Energy storage from conducting polymer

(Yu et al., 2011). The film of PEDOT:PSS decreases graphene/MnO2 composite’s internal resistivity. CP film of PEDOT:PSS serves as binder materials conductive additives as well as binder materials. Redox reactionbased pseudocapacitance helps it to participate in the charge storing mechanism. Coating of the polymer intercepts disintegration and fragmentation of MnO2 and suspension of ions during continuous chargingdischarging mechanism. In recent times, cable structured flexible supercapacitors evolved from three-dimensional CNT-cotton thread/ MnO2/PPy composited electrodes are fabricated by a simple three-phase mechanism, where first ink of single-walled CNT is layered on the surface of cotton threads and then PPy films and MnO2 nanomaterials are easily grown on the cotton threads layered with CNT. This is done by in situ electrochemical deposition mechanism. Compared to carbonaceous components, transition metal hydroxides/oxides are able to deliver high specific capacitance according to reversible and fast redox reactions. However, comparatively poor cycling stability and low power density make them inappropriate for practical applications. They have lower power density because of inferior current conducting nature of the metal compounds for confining speedy electron transportation. Poor cycling stability makes structure of electrode materials weaker. Hence, it can be smoothly impaired by protrusion and diminution during chargingdischarging mechanism. Conversely, CPs have comparatively higher current conducting capacity. Hence the acting synergic effect between metal oxides/hydroxides and CPs enhances electrochemical characteristics. In these types of composites, the oxide/hydroxide of metals generally impart contribution in increasing specific capacitance based on morphological characteristics and redox properties. The CPs are pivotal for higher electrical conductivities and higher mechanical stability facilitating rapid transportation of electron inside the composite material for higher chargedischarge rate. Their electrochemical properties are significantly influenced by crystallinity of metal oxide/hydroxide, morphology, or surface area (Yan et al., 2014; Wei, Cui, Chen, & Ivey, 2011). Other low-cost transition oxides/hydroxides of metals, for example, Fe2O3, TiO2, NiO/Ni(OH)2, MnO2, and Co3O4/Co(OH)2 are used. Generally, core/shell coaxially aligned nanorods/wires are synthesized by two strategies. In the first step, one-dimensional metal hydroxide/oxide is prepared that acts as core and then it is coated with a coating of CPs in the form of shell via electrochemical or chemical processes. Various types of coaxially aligned metal compound/composites of CPs, for example, CoO/PPy, MnO2/PPy, and MnO2/PEDOT are effectively synthesized and exhibit superior mechanical as well as superior electrochemical characteristics (Liu & Sang, 2008; Xie et al., 2011; Yao, Zhou, & Lu, 2013; Zhou, Zhang, Li, & Liu, 2013). For instance, nanowire array of CoO combined with PPy exhibits specific capacitance extraordinarily as high (2223 F/g), and 99.8% cycling stability (Zhou et al., 2013). Mainly, the core of CoO attributes to higher energy storing capacity; on the other hand, PPy shell which is flexible, porous, and the highly conductive facilitates bothion diffusion and transportation of electron inside the core of CoO as well as protects the core from disintegrating and



CHAPTER 11 Appraisal of conducting polymers

shattering during chargedischarge mechanism. But the only disadvantage is the CP prevents the exposure of electrolyte with the core of metal oxide core and thus the energy density suffers. Metaloorganic frameworks (MOFs) are a novel set of porous materials, famous for having high porosity and high specific areas. Hence these materials can be used in drug delivery, sensor, catalysis, and gas storage and separation (Furukawa, 1989). They are also employed as electrode material in supercapacitors due to improved specific surface areas and exceptional porosities that increase availability of ions by diffusion effect in MOFs electrode. Generally, supercapacitors made up of MOF are divided into two categories: (Granero et al., 2011) preparation of porous carbon or metal oxide where MOFs act as original template, and (Weng et al., 2015) for energy storing purposes by electric double layer capacitor (EDLCs) method or through redox reaction of pseudocapacitor of metal centers pure MOFs are employed as novel electrode materials (Liu, Shioyama, Jiang, Zhang, & Xu, 2010; Yang, Xiong, Zheng, Qiu, & Wei, 2014). Supercapacitor’s effectiveness is hugely regulated by the electrode materials and the electrolyte used. Energy and power density of any device proportionally change with square of operating voltage. Typically, three varieties of the liquid electrolytes are employed in supercapacitors—ionic liquids (ILs), aqueous, and organic electrolytes. Aqueous electrolytes are easy to fabricate, possess high ionic conductivity, high safety, low viscosity, nonflammability, and cost-effectiveness are the major advantages of aqueous electrolytes. Their voltage limit is B1.0 V which is lower compared to organic electrolytes having voltage window of 2.53 V. Thus energy density of organic electrolytes is 614 times higher than aqueous electrolyte. IL, organic solutions, and aqueous solutions based on traditional supercapacitors are not suitable as flexible and portable electronics. To overcome these problems, solid-state supercapacitors are applied for energy storing. In comparison with liquid capacitors, solid-state supercapacitors share merits like flexibility, high safety and lightweight (Wang et al., 2014). The solid-state electrolyte acts as the electrode divider and ionic conducting media. It has characteristics (Gao & Lian, 2014) such as high electrochemical stability, wide voltage window, stability, good formability, greater mechanical strength, low electrical conductivity at room temperatures, and higher ionic conductivity (Lu, Yu, Wang, Tong, & Li, 2014; Zhong et al., 2015). Polymer gel electrolytes exhibit comparatively higher ionic conductivity so they are the most commonly used solid-state electrolytes. They are composed of polymeric frameworks which act as host material providing superior mechanical integrity. The electrolytic salts provide ionic conduction and aqueous or organic solvent impart ion-conducting medium. The most commonly used host polymers are potassium polyacrylate, poly(acrylic acid), etc. In supercapacitors, directly polymeric materials are used as substrates. For fabricating flexible supercapacitors, polyethylene terephthalate (PET) and PDMS are widely employed as substrates. PDMS films are used to prepare translucent and flexible thin filmed supercapacitor. For increasing thermal, electronic, and mechanical stability of PDMS films, single-walled CNTs are introduced (Yuksel, Sarioba, Cirpan,

11.3 Energy storage from conducting polymer

Hiralal, & Unalan, 2014). For achieving high electrical conductivity and flexibility, conducting materials like CNT and graphene are deposited onto PET films. Other polymeric materials are also applied apart from PET and PDMS. Localized pulsed laser radiation is applied for rapid transformation of pure polyimide (PI) surface into electrical conductive porous structure made up of carbon. Using programmable laser scanning method, the interdigitated pattern of electrode is grown on PI sheets. To fabricate conducting flexible electrodes for preparing flexible supercapacitor, the PI sheets work as precursor for carbonization and a flexible substrate according to this method. This consolidated electrode exhibits better flexibility and stability in comparison with conducting material-coated polymeric substrate-based electrodes. Pure polymeric materials-like nonwoven cloths synthesized from papers and wood fibers are used as substrates that are lightweight and flexible. Flexible CNT/paper electrodes are synthesized by coating a paper with CNTs. In LIB, cathode and anode are isolated by ion conducting electrolytic solution (Lee, Yanilmaz, Toprakci, Fu, & Zhang, 2014). Reversible electrochemical redox reactions procure the capacity of LIB between two electrodes. Lithium ions travel from cathode to electrolyte and then they reach the surface of anode during charging mechanism. Subsequently, electrons move cathode to anode through external circuitry. This whole mechanism is altered in discharge process. Electrodes are composed of materials such as binders, conducting additives, and active materials and they are connected with current collectors. Separator is positioned between cathode and anode to tackle short circuit. The separator also absorbs electrolyte. Polymers have a significant role in LIB fabrication. The parameters of LIBs such as chargingdischarging rate, perpetuity, capacity, voltage basically are dependent on active materials of anode and cathode. The mostly employed materials for cathode are LiFePO4, LiMn2O4, Li4Ti5O12, and LiCoO2. Subsequently, the materials used for anode are Sn, Si, and graphite. The conventional inorganic cathode materials suffer from poor structure stability and low theoretical specific capacity. Meanwhile, due to possible environmental pollution and limited resources, the massive scale fabrication of cathode materials based on transition metal is hampered. Polymer-based electrode provides greater theoretical capacities compared to inorganic materials. On the other hand, organic polymer electrodes exhibit higher power densities and higher rate performance compared to inorganic-based electrode due to rapid organic redox reactions. So, for replacing inorganic electrode materials, extensive studies are going on to explore polymeric electrode materials. The five major classes of conductive polymers are polythiophene (PTh), polyaniline (PAn), polyparaphenylene (PPP), and polyacetylene (Pac). In the past decades, these are widely explored as electrode constituents. In case of organosulfides, the SS bond can be fragmented and reconstructed in reversible manner. In comparison with the conductive polymers, superior performance can be attained by the redox reaction. Hence, for rechargeable LIBs, organosulfides are acknowledged as preferable organic constituents. A typical example of organosulfides is PDMcT. Organic bipolar polymers of nitroxyl radical are employed as p-type polymers to achieve stable cycling performance



CHAPTER 11 Appraisal of conducting polymers

and higher discharge voltage. Among them, the most widely used material is PTMA [poly(2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl methacrylate)]. Polymers of nitroxyl radical have fast reaction kinetics which is favorable for LIBs. Due to properties such as high structural diversity, fast reaction kinetics and high theorical capacity, conjugated carbonyl polymer is considered preferable organic component for LIB. The electrochemical redox reaction is simplified as a reverse reaction of the carbonyl group and an enolization reaction. For promoting the reaction, conjugated structure is crucial. Electroactive polymer materials have several merits, for example, good flexibility, good design versatility, and high capacity. But they are restrained from practical applications due to few internal limitations. Subsequently, other than CPs, polymer electrode constituents have limitations like poor electrical conductivity. During the reaction of electrode, this causes slowdown of transportation of electron. Hence, extra efforts are given to overcome this drawback and to improve electrochemical performances. One method is conductive carbon materials; for example, porous carbon, graphene, carbon fiber (CF), and CNT are coupled with the polymer and a composite electrode is formed. This removes major drawbacks. Due to properties of CFs such as chemical resistance, thermal and electrical conductivity, and high strength, they have been on focus of attraction. The fusion of polymer electrode constituents and CFs improves the electronic conductivity of insulated polymer. Following this method, the rate capability and cyclability are observed to be exceptionally upgraded. Since CNT has unparallel chemical and physical characteristics like high mechanical strength, good chemical stability, and high electrical conductivity, it is assumed as suitable constituent used for LIBs. CNTs are integrated into polymeric components which result in the formation of hybrid electrodes. Capacity of LIBs can be improved by incorporating conducting additives like CNTs into the hybrid electrodes. At the 80th cycle the resultant LIB exhibits discharge capacity of 86 mAh/g (Sivakkumar and Kim, 2007). At twice rate, it exhibits remarkable discharge capacity of 65 mAh/g. This value is higher compared to what is achieved by the combination of super-p carbon and polyaniline. Graphene shows extraordinary chemical and physical properties, for example, high stability, mechanical strength, huge theoretical specific surface area, and remarkable electrical conductivity. Hence, it is evaluated to be a potential candidate as polymeric electrode material for LIBs. For instance, via dispersiondeposition mechanism, graphene is introduced into PTMA (Guo, Yin, Xin, Guo, & Wan, 2012). By in situ polymerization method, polymer/graphene hybrid is manufactured with highly loaded active materials. Using this strategy poly(anthraquinonyl sulfide)/ graphene can be successfully prepared (Song et al., 2012). In comparison with pure polymers, nanocomposite exhibits six times higher electrical conductivity because graphene has high dispersion capability. Interlinked nanochannels present in the porous carbon materials enfold redox-active electrode constituents. They enhance the properties of polymeric electrodes majorly in two methods. In one method, the nanochannels provide accommodation to the polymers, facilitation of the electrolytic infusion, and prevention of the dissolution mechanism. In the

11.4 Energy harvesting based on polymer

second method the structure of carbon matrix enhances rapid flow of electron. But, the size of pore diameter impacts the performance of the organic-carbon hybrid. Larger pore size enables the filling of molecules.

11.4 Energy harvesting based on polymer We have limited reserves of fossil fuels. To fulfill ever-expanding need of electricity, we need to find out nonconventional sources (Cook et al., 2010; Dillon, 2010). New devices are continuously invented to harvest ambient energy in the form of heat, mechanical vibration, and light (Zeng et al., 2014). Polymers are strong, lightweight, characteristic-tunable, solution-processable material. Polymers are promising candidate for developing flexible, low cost, harvesting devices at large scale (Coakley & McGehee, 2004). Photovoltaic devices are capable to generate electric current or voltage in the presence of light and convert photo energy into electricity (Tang, 1986). Polymer or plastic solar cell having high efficiency of energy conversion (Dun et al., 2015) is used in the applications of photovoltaic devices. In a photovoltaic process, excitons are generated and free carriers are produced from incident photons absorbed by conjugated polymers. Excitons are nothing but bonded electronhole pairs that dissociate and free carriers formed at interface of donor or acceptor and migrate into transporting materials. Afterwards, electrons are deposited in anode and holes are collected in cathode. The electrodes are present after the electron and hole extraction layer. Electronsholes are then transferred to external circuit for the formation electricity from light (Zhu, Yang, & Muntwiler, 2009; Collavini, Vo¨lker, & Delgado, 2015). The solar cell’s performance is determined by fill factor connected with microscopic morphology of charge extraction layer, active layer, and treatment done after deposition process (Guo et al., 2013). For enhancing the photovoltaic performances of solar cell, fill factor (voltage, open-, and short-circuit current) must be upgraded. Typically, fabricated conjugated polymer is employed as donor, which is also p-type semiconductor. Polymer mixed with [6,6]-phenylC61-butyric acid methyl ester (PCBM) is used as acceptor and light-harvesting polymer is used as a donor for absorbing light as excitation source. The most popular conjugated polymers are derivatives of polythiophene (PTh), such as poly(3hexylthiphene) (P3HT). Maximum efficiency of 5% can be observed when PCBM is employed as acceptor and P3HT is used as donor. For electron injection and transport in heterojunction blend, another important component is acceptor components which are derivatives of fullerene especially PCBM and PCBM (Wienk et al., 2003). Recently, n-type conjugated polymers that produce every polymer solar cell (PSC) are developed as acceptor materials (Facchetti, 2013). The morphology basically implies the phase segregation and crystallite of conjugated polymers in heterojunction mixture. In dye sensitized solar cells (DSSC),



CHAPTER 11 Appraisal of conducting polymers

different types of conducting polymers such as polypyrrole (PPy), PEDOT, PANI are used as counter electrode. Generally, PEDOT:PSS, which is a p-type conducting polymer, is used in solar cells as hole-transporting element. To eliminate the drawback of traditional DSSC being highly volatile, several p-type conductors or solid-state electrolytes are being realized for solid-state DSSCs. Piezoelectric generator or transducer transforms vibrational energy of the particles present in the material into electrical energy for driving minute devices that require less energy and can run for a long time. Piezoelectric components that are interposed between two electrodes can induce strain which creates electric field and thus voltage after applying an external force. Some polymer materials such as polyuria (Hattori, Takahashi, Iijima, & Fukada, 1996), nylon-11 (Newman, Chen, Pae, & Scheinbeim, 1980), and PVDF and its copolymer (Furukawa, 1989) are used to fabricate most of the polymer-based piezoelectric generators. Similarly, triboelectric effect is defined as the charge transfer between any two materials. Triggering potential for triboelectrification and preserving the induced charge on the surface of the dielectric is assumed as parameters in case of triboelectric generator. According to the electrostatic effect and contact triboelectrification, based on specified applications, various generator structures can be designed. Generally, these generator structures are classified into four groups—(Wang, Lin, & Wang, 2015) (1) contact mode (Fan, Tian, & Lin Wang, 2012), (2) single mode, (3) sliding mode (Li et al., 2015; Niu et al., 2013), and (4) freestanding triboelectric-layer mode structure (Niu et al., 2015). Efficiency of triboelectric generator depends on the surface area of triboelectrified materials. Typically, PDMS, Kapton (polyimide), and polytetrafluoroethylene (PTFE) are used as dielectric materials. Triboelectric generator is applied for converting mechanical energy into electric signal via electrostatic induction and triboelectrification and this process is quite similar to piezoelectric generator. For example, blending with polyvinylidene fluoride can also be explored to develop flexible devices based on conducting polymers (Sengupta et al., 2021; Sengupta, Ghosh, Bose, Mukherjee, Roy Chowdhury, Datta), as can be observed in Fig. 11.3. Another class of energy harvesting devices are thermoelectric generators that depict a different method of harvesting energy and generating electricity. Inorganic elements are generally applied in thermoelectric generators. But inorganic components are heavyweight and fragile in nature and they have high annealing temperature. As illustrated in Fig. 11.4, highly efficient, lightweight polymer materials are used as thermoelectric generators (Zhang et al., 2021). In the 1820s, thermoelectric generator, based on Seebeck effect, was innovated. Diffusion of carriers like electrons or holes generates diffusing potential which further resists diffusion via drift current. Eventually, an equilibrium is established between drift current and diffusion current, creating a temperaturedependent electric field at this junction. It is used to measure temperature. The ample availability of sunlight as thermal energy and excessive wastage of heat energy from automobiles proves that thermoelectric generators can be successfully used as low-cost energy harvesting sources. To calculate the efficiency of thermodynamic generator, the output power (Pout) is divided by the input heat

11.4 Energy harvesting based on polymer

FIGURE 11.3 A flexible piezoelectric nanogenerator fabricated using electrospinning technique of conducting polymers depicting human motion energy harvesting from (A) heel, toe movement, and wrist movement, and (B) finger twisting movements (Sengupta, Das, Dasgupta, Sengupta, & Datta, 2021). Reproduced with permission from Sengupta, A., Das, S., Dasgupta, S., Sengupta, P., Datta, P. (2021). Flexible Nanogenerator from electrospun PVDFpolycarbazole nanofiber membranes for human motion energy-harvesting device applications. ACS Biomaterials Science and Engineering, 7(4),16731685.

(Qin), so the efficiency (η) is formulated as η 5 Pout/Qin. Power conversion efficacy of thermodynamic generator can be calculated by using following formula (Culebras, Uriol, Go´mez, & Cantarero, 2015): pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 1 ZTav 2 1 ηmax 5 Φc pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  ΦC γ 1 1 ZTav 1 Th =Tc



CHAPTER 11 Appraisal of conducting polymers

FIGURE 11.4 Representative 3D printing of flexible electrodes composed of conducting polymers: (A) design wearable thermocell, (B) fabrication of wearable thermocell, (C) flexible strapshaped thermocell, and (D) strap-shaped thermocell charge supercapacitor and light a lab timer. Reproduced from Zhang, S., Zhou, Y., Liu, Y., Wallace, G.G., Beirne, S., & Chen, J. (2021). All-polymer wearable thermoelectrochemical cells harvesting body heat. iScience, 24(12), 103466.

where Tc and Th represent the low and high temperature, respectively. Meantime, Tav is the mean of low and high temperature and ΦC 5 (Th 2 Tc)/Th is Carnot efficiency. Z is figure of merit and it is calculated as: ZT 5

α2 σ T κ

where α is the Seebeck coefficient, k is thermal conductivity of material, σ is the electrical conductivity. For enhancing efficiency of thermoelectric generators, Seebeck coefficient and electrical conductivity are concurrently increased with constant conductivity. In thermoelectric generators, p-type conductive polymers are employed. The mostly used conducting polymers in thermoelectricity are PANI, PPy, polyalkylthiophenes and its derivatives, polyacetylene, PEDOT and poly(2,7-carbazolyenevinylene) (Wei, Mukaida, Kirihara, Naitoh, & Ishida, 2015). Changing parameters

11.5 Organic light-emitting diodes

such as backbone structure and side-chain length, electrical behavior of semiconductive conjugated polymer can be effectively regulated. Electrical conductivity and Seebeck coefficient are affected by doping level of the constituents (Mengistie et al., 2015). The polymer conformation and morphology influences its electrical conductivity. By changing polymerization parameters such as electrolytes, temperature, current, density and monomer concentration, thermoelectric property can be modulated. It has been found out that various types of charge carriers influence the thermoelectric behavior of conductive polymer materials (Wang, Ail, Gabrielsson, Berggren, & Crispin, 2015).

11.5 Organic light-emitting diodes The phenomenon by which the electrical excitation causes emission of light energy is called electroluminescence. First time, polymer light-emitting diodes (LEDs) were successfully developed using PET by Heeger and his coworkers in 1992. This innovation made lead to creation of flexible display devices. Later in 1994, polymer-based white light-emitting electroluminescent device was developed by Matsumoto and Kido (Kido, Hongawa, Okuyama, & Nagai, 1994). In comparison with inorganic LEDs, polymer light emitting diodes (PLEDs) inherit various advantageous properties such as high flexibility, adaptability for large area fabrication, compatibility to solution process, low cost, and numerous applicable materials. The electrons and holes overcome the threshold of the interface when the bias voltage is exerted externally across the device. The electrons leave the cathode and enter the organic layer. Similarly, holes travel from anode to organic layer. Electrons flow through the lowest unoccupied molecular orbital (LUMO) region which is a part of the electron transporting layer (ETL) and holes flow through the highest occupied molecular orbital (HOMO) region present in the hole transporting layer (HTL). The external electric field excites the carriers and they move to the emission layer. There is emission of light as soon as the excitons bounce back to ground state. Color of emitted light depends on variation in energy between HOMO and LUMO regions of organic material. For achieving high luminous efficiency and low driving voltage, the effectiveness of injection and transportation of charge carriers are important. For achieving lower driving voltage, two factors are important. During the charge carrier injection mechanism, Ohmic contact between the organic layers and the electrodes is the first factor and during the charge carrier transportation mechanism, maximizing the mobility of carriers is the second important factor. Applied bias voltage when overcomes the interfacial gap between organic material and electrode, the charge carriers are injected. The minimum voltage applied for exceeding the interfacial gap is known as turn-on voltage. Lowering the interfacial gap increases light-emitting efficiency by incorporating buffer layers. The mobility of charge carriers of organic molecular materials is comparatively low (Campbell Scott & Malliaras, 1999). The



CHAPTER 11 Appraisal of conducting polymers

electrons are confined inside single molecules. Consequently, PLEDs do not have high luminous efficiency and low turn-on voltage because injection and transportation of charge carriers act as limiting factors. Rate of injection at an electrode having limited contact is proportional to the mobility of charge carriers of organic materials. Similarly to improve charge carrier transport, ETL and HTL are introduced for realizing successful transportation of electrons and holes simultaneously. It is demonstrated that introducing a PPV layer between emissive PF layer and PEDOT:PSS to obstruct electrons results in increasing the maximum luminous efficiency twice. Polymerized perfluorocyclobutane (PFCB)-based copolymers consist of electron-rich triarylamine side chains which help in transportation of blocking electrons and holes. Amongst various types of copolymers, bis(N,N0 -diphenyl-N,N0 bis(3-butylphenyl)-(1,10 -biphenyl)-4,40 amine)-PFCB (BTPD-PFCB) has maximum capability to transport holes compared to PEDOT:PSS and the maximum triarylamine group density. Work function of BTPD-PFCB and PEDOT:PSS are 5.3 eV and 5.2 eV, respectively. Hence, for emissive materials having comparatively poor HOMO level, the number of holes injected from HTL and released in the emission layer is relatively increased. In comparison, with PEDOT:PSS, copolymers based on PFCB when employed as HTL demonstrate superior electron blocking and hole transporting characteristics. But the fabrication method of PFCB films is very complex. In place of PEDOT:PSS, thin metal oxide films like V2O5, MoO3, WO3 can be used due to several advantages. However, vacuum evaporation technique is followed for preparing the thin metal oxide films, which restricts their usage in PLED. Carbon-based materials have several applications because of improved mechanical flexibility, conductivity, and optical transmittance. Graphene oxide (GO) is an another material which can be employed as alternate of PEDOT:PSS to make HTL. As the middle layer composed of GO prevents movement of electrons from emission layer to indium tin oxide (ITO) and thus the charge carrier injection balance is improved and simultaneously avoids radiative quenching of excitons (Lee et al., 2012). When the performances of PLED with various types of HTL layers are analyzed in the form of power efficiency voltage (P-V), luminous efficiency voltage (E-V), L-V, and J-V curves, it is seen that for those PLEDs where PEDOT:PSS is used as HTL, at 12.6 V the highest luminance is 33,800 cd/m2 and at 9.6 V the highest luminous efficiency is 8.7 cd/A. However, in case of those PLEDs where rGO is employed as HTL, it is observed that at 13.0 V the highest luminance is 8300 cd/m2 and at 8.6 V the highest luminous efficiency is 5.0 cd/A. This indicates that when rGO is used as HTL, both luminance and luminous efficiency decrease compared to those with PEDOT:PSS since rGO has poor hole injection capability and electrical conductivity due to greater interfacial contact gap between HTL and emission layer. When GO is used as HTL, the properties of PLED are dependent on width of the GO films. The GO film’s width is 4.3 nm for the optimized PLEDs. In this case it is observed that at 4.4 V the highest power efficiency is 11.0 lm/W, at 6.8 V the highest luminous efficiency is 19.1 cd/A and at 10.8 V the highest luminance is 39,000 cd/m2, which are boosted by 280%, 220%, and 120%, respectively, in comparison with standard PLEDs where PEDOT:PSS is used HTL.

11.6 Electrochromic materials and devices

GO has better capability to block electrons to itself due to their large bandgap. The probability of electron hole recombination is enhanced by this property. Hence such high performance is obtained. Thinner GO films make it tough for the material for realizing full coverage. This is not good for properties to block electrons. The electrical conductivity increases in case of thicker GO films and it leaves poor impact on hole transportation mechanism. An example of application of conducting polymers as organic LEDs based on PEDOT polymer treated with benzoic acid (Kang, Kim, & Kim, 2021) and their characterization is shown in Fig. 11.5. Electron transporting or injection layers (ETL) are used in PLED for decreasing the barrier for electron injection between emission layer and cathode, implying that ETL should have good electron affinity and high ionization potential for blocking hole. Other than inorganic material and tiny organic molecules, conjugated polyelectrolytes (CPEs) effectively reduce the electron junction barrier. CPEs are more preferred in practical applications as it removes metals having lower work function like Ba and Ca, which are highly sensitive to surrounding atmosphere, decreasing the device’s longevity. Using polyfluorene backbone, poly(9,9-bis(2-(2-ethoxyethoxy)ethyl)fluorene) uniformly forms film outside the emission layer. This is a type of neutral CPE. In the form of cathode modifiers, anionic and cationic CPEs are also employed. After applying external voltage, counterions migrate to the interfacial plane existing between CPEs and emission layer in case of PLEDs where cationic CPEs are employed as ETL. Luminous efficiency of the PLED decreases due to the quenching effect of counterions. In case of PLEDs, where ETL is made up of anionic CPEs, the mobile counterions transfer to the cathode for obtaining strongly aligned dipole. Hence, a bilayered structure is created between the cathode and the mobile ions facilitating injection of electrons. As cathode modifier, anionic CPEs are relatively more appropriate than cationic CPEs. Compared to the traditional design, the inverted design eliminates the materials which inject electrons and are air-sensitive like Ca, for solving difficulties of deterioration of the device at normal room temperature. For electron injection layer present in the inverted PLED, air-stable metal oxides are generally employed. ZnO is highly preferred material amongst them since they have n-type characteristics which originate from oxygen vacancies and interstitial Zn (Bolink, Coronado, Repetto, & Sessolo, 2007). Branched polyethyleneimine ethoxylated (PEIE) and polyethyleneimine (PEI) act as layer for injecting electrons in invertedly designed PLEDs (Kim et al., 2014). PEIE and PEI are air stable and consist of amine groups which form string of dipoles between the ZnO surface and PEI. These strong dipoles are favorable for decreasing the gap of electron injection and improving its mechanism.

11.6 Electrochromic materials and devices Electrochromism is the event of reversibly changing colors shown by some materials. For fabricating electrochromic devices, several materials are employed, for example, polymers, photonic crystals, liquid crystals, and transition metal oxides



CHAPTER 11 Appraisal of conducting polymers

FIGURE 11.5 Luminescence and current-density properties of organic light-emitting diodes composed of conducting polymers depicted by (A) schematic representation of bending test, voltage vs (B) current density and (C) luminance, (D) current efficiency relationship with current density, and (E) performance stability under bending test. Reproduced from Kang, H.S., Kim, D.H., Kim, T.W. (2021). Organic light-emitting devices based on conducting polymer treated with benzoic acid. Scientific reports, 11(1), 3885.

(Arsenault, Puzzo, Manners, & Ozin, 2007; Booth & Casey, 2009; Nicoletta et al., 2005). Various procedures describe the color-changing property of polymeric electrochromic material such as electrothermal chromatic transition and electro-induced oxidation reduction.

11.6 Electrochromic materials and devices

Conjugated polymers have some distinctive characteristics like color versatility and electrical conductivity and employed as ideal electrochromic materials especially in textiles, displayers, mirrors, and sensors. Under a specific voltage the chemical species reversible changes between two redox states are responsible for their reversible electrochromic behaviors. This process is called electroinduced oxidation reduction (Beaujuge, Amb, & Reynolds, 2010; Niklasson & Granqvist, 2007). Based on this, the multilayered structure of an electrochromic device comprises ion storage/electrochromic layer, electrolyte, electrochromic, substrate, and transparent conductor (Thakur, Ding, Ma, Lee, & Lu, 2012). The translucent conductor as the functional electrode adheres to electrochromic layer; electrolyte serves in ions conduction and supports redox reaction. As soon as ions are introduced in electrochromic layer, ion storing layer acts as a buffer which captures the ions from electrolytic layer. With advancement in electrochromic devices, another electrochromic layer replaces the ion storing layer. Electrochromic materials and their derivatives (namely, PPy, PANI, PTh) are mostly explored conjugated polymers. Among them, first conjugated polymer was PTh to be used as electrochromic material (Garnier, Tourillon, Gazard, & Dubois, 1983). But, the insoluble nature of PTh makes it difficult to synthesize its derivatives. Poly(3,4-ethylenedioxythiophene) (PEDOT) derived from PTh has unique electro-optical properties, high stability, and good conductivity. Exhibiting properties such as rapid switching of color, environmental stability, and low cost, PANI is another promising conjugated polymer. Hence, PANI has high potential for practical application. If the applied voltage is increased, PANI exhibits a moderate change in colors among black, blue, green, and yellow along with high reversibility and swift response. The conventional electrochromic materials can create small range of colors. To reinforce the change in colors, other electrochromic polymers are fabricated displaying purple (Reeves et al., 2004), blue (Balan, Gunbas, Durmus, & Toppare, 2008; Invernale et al., 2009; Wu, Lu, Chang, & Wei, 2007), green (Durmus, Gunbas, Camurlu, & Toppare, 2007; Gunbas, Durmus, & Toppare, 2008), yellow (Lin et al., 2015), red (Dyer, Craig, Babiarz, Kiyak, & Reynolds, 2010), and orange (Dyer et al., 2010). Conjugated polymers which can produce various colors at neutral, intermediate, and doped states are prepared (Thompson, Schottland, Zong, & Reynolds, 2000). Changing the components of monomers, the colors of copolymers are easily readily regulated (Beaujuge, Ellinger, & Reynolds, 2008). The π-conjugated polymers such as poly (3-methylthiophene) (P3MT), poly(2,5-dimethoxyaniline) (PDMA), and PEDOT are introduced into the wires made up of stainless steel for preparing electrochromic fibers by electrochemical polymerization method. After lamination of gel on electrochromic layer, stainless steel wire is crumpled. The electrochromic fibers show reversible and rapid change in colors (Li, Zhang, Wang, & Li, 2014). One of the favorable strategies is to prepare polymeric or inorganic hybrid materials. NiO/PANI (Sonavane, Inamdar, Deshmukh, & Patil, 2010), NiO/PEDOT (Xia et al., 2009), NiO/PPy (Sonavane, Inamdar, Dalavi, Deshmukh, & Patil, 2010), PEDOT/Au/CdSe (Bhandari et al., 2010), WO3/PANI (Ma, Shi, Wang, Zhang, &



CHAPTER 11 Appraisal of conducting polymers

Li, 2014), PANI/Graphene (Sheng, Bai, Sun, Li, & Shi, 2011), and PEDOT/CNT (Bhandari, Deepa, Srivastava, Lal, & Kant, 2008) are few examples of inorganic hybrid or polymeric materials having exceptional electrochromic characteristics. Synergetic characteristics of individual constituents help these hybrid materials to exhibit high coloration efficiency and fast responsiveness. Polymer dispersed liquid crystals (PDLCs) are micrometer-sized crystalline droplets of liquid scattered into the polymeric matrix. Under electric field, they are able to convert themselves into opaque states from transparent states. The liquid crystal molecules are realigned by the electric field and transmittance changes reversibly. Functional device comprising thermoplastic matrices and crystalline nematic liquids, which can change its colors by electricity are developed from PDLC film (Nicoletta et al., 2005). Due to scattering of light by crystalline liquids dispersed into the polymeric matrix, these electrochromic devices are not transparent. After applying an electronic field of 1 V/μm, the crystalline liquids reconstruct themselves along the electric field at droplet surface and thus increasing transmittance to 8% from 2%. The liquid crystals retain its initial random arrangement as soon as the electric field is removed. Due to unique electrochromic characteristics, photonic crystals are widely explored. Photonic crystals inherit properties such as metallodielectric, supercapacitor structures which control propagation of electromagnetic waves setting a definition of forbidden and allow photonic energy bands. According to Bragg’s law, they show functions such as controlling photonic movement, reflecting thin wavelength bands, and exhibiting compositional colors. Since photonic crystals have exceptional properties such as highly aligned structures, multiresponsive colors, and nonbleachable compositional colors which cover the complete visible range of spectrum. According to volume variation stimulated by temperature and electro-induced redox reactions, the lattice constants present in the crystal structures change and thus color transition takes place in photonic crystals (Seeboth, Lo¨tzsch, Ruhmann, & Muehling, 2014). The PSCs possess characteristics features like flexibility, facile association with other devices, and lightweight. Plastic substrate PET films coated with ITO are used for fabrication of flexible polymeric solar cells. For fabricating flexible electrodes, as a replacement of ITO layer, conductive polymers are directly used to laminate polymeric substrates while preparing flexible electrodes. To increase conductivity of the electrodes, varieties of metal grids other than conductive polymers are employed for constructing composite layer. Silver ink is deposited on PET surface to manufacture flexible ITO free conducting PET film. For instance, flexible electrodes exhibit 4.8% of maximum power conversion efficiency and high flexibility (Nickel et al., 2014). In construction process of PSCs, polymers are also employed as working layers, other than acting as conducting layer and substrate. Flexible energy storing devices gain a lot of attraction. Selecting appropriate electrode material and designing suitable structure for achieving stable electrochemical and mechanical performances during stretching, twisting or bending are quite


challenging. Introducing polymers or polymeric composites for flexibility enhancement is considered one efficient strategy. At academic or industrial level, supercapacitors and LIBs are widely explored.

11.7 Conclusions Conducting polymers have immense potential in the fields of sustainable and flexible electronics, flexible devices, prosthetics and drug delivery devices, corrosionresistant coatings, and biosensing applications. An extensive understanding of the mechanism of electrical conduction and doping effects is allowing the synthesis of new conducting polymers as well as adoption of surface modification techniques to functionalize biomolecules on the conducting polymers. However, developments in increasing the solubility of these polymers will be one of the key aspects to enhance their industrial applicability and processing. Similarly, stability of conducting polymers is another area which needs to be improved for wider applications. For, life cycle of supercapacitors based on conducting polymers is an area seeking improvements. Moreover, biomedical applications of conductive polymers are yet another vast unexplored area. It is imperative that researches into new conducting polymers and composites are enhanced intensively in the coming years.

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Zhang, X., Pint, C. L., Lee, M. H., Schubert, B. E., Jamshidi, A., Takei, K., et al. (2011). Optically- and thermally-responsive programmable materials based on carbon nanotube-hydrogel polymer composites. Nano Letters, 11(8), 32393244. Zhang, X., Yu, Z., Wang, C., Zarrouk, D., Seo, J. W. T., Cheng, J. C., et al. (2014). Photoactuators and motors based on carbon nanotubes with selective chirality distributions. Nature communications, 5. Zhao, X., & Wang, Q. (2014). Harnessing large deformation and instabilities of soft dielectrics: Theory, experiment, and application. Applied Physics Reviews, 1, 021304. Zhong, C., Deng, Y., Hu, W., Qiao, J., Zhang, L., & Zhang, J. (2015). A review of electrolyte materials and compositions for electrochemical supercapacitors. Chemical Society Reviews, 44, 74847539. Zhou, C., Zhang, Y., Li, Y., & Liu, J. (2013). Construction of high-capacitance 3D CoO@Polypyrrole nanowire array electrode for aqueous asymmetric supercapacitor. Nano Letters, 13(5), 20782085. Zhu, X. Y., Yang, Q., & Muntwiler, M. (2009). Charge-transfer excitons at organic semiconductor surfaces and interfaces. Accounts of Chemical Research, 42(11), 17791787. Zhu, Y., Murali, S., Stoller, M. D., Ganesh, K. J., Cai, W., Ferreira, P. J., et al. (2011). Carbon-based supercapacitors produced by activation of graphene. Science, 332, 15371541.


Shape-memory polymers

12 Deepshikha Rathore

Amity School of Applied Sciences, Amity University Rajasthan, Jaipur, Rajasthan, India

12.1 Introduction Shape-memory polymers (SMPs) are an incipient category of intellectual polymers, because on suitable stimulation they are capable to alter their shape in a previous manner. SMPs can attain temporary shape in the form of deformation, which is identified as second fixed shape, until deformed system is exposed to a suitable stimulus. After imposing appropriate stimulus, system gains its original shape immediately. As such polymers remember their memorized shape, hence they are known as SMPs (Lendlein, 2010). Those stimuli reactive polymers can transform their mechanical, optical, and electrical properties substantially, including shape, phase separation, surface, permeability upon slight change of atmospheric conditions such as pH, temperature, magnetic field, light, electric field, sonic field, ions, solvent, glucose, and enzyme (Meng & Li, 2013). These transformations on demand are of scientific and technological importance and can be explained in three significant processes as shown in Fig. 12.1: 1. Programming: It is the first step, in which, on applying mechanical force at eminent temperature, SMPs hold impermanent shape after changing their previous shape.

FIGURE 12.1 Schematic representation of the thermally induced one-way SME. SME, Shape-memory effect. Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 12 Shape-memory polymers

2. Storage: It is the second step, in which, on cooling under stimulation temperature and removing applied mechanical force, impermanent shape of SMPs is sealed. 3. Recovery: It is the third step, in which, SMPs achieve their previous shape.

12.2 Various shape-memory polymers A variety of SMPs have been developed, in which three very common polymers (polylactide, polytetrafluoroethylene, and ethylene-vinyl acetate) were used. Those polymers exhibit shape-memory effect (SME), which are categorized in cross-linking and thermal transitions to understand their comprehensive features (Safranski & Griffis, 2017).

12.2.1 Cross-linking The various polymers are available, including several types of structures. Those structures are (1) linear, (2) branched, and (3) linked in the form of network, which depends upon the variety of monomers utilized in formation of polymers and shown in Fig. 12.2. To form the polymer network, polymer chains are linked together either by covalent bond or by physical entanglements. This linkage process restricts permanent chain dislocation and prevents chain’s mobility, due to which netpoints generate, which are permanent. Hence, the original shape establishes for the SMP. This cross-linking of molecule or polymer chain can be

FIGURE 12.2 Schematic of polymer structures: (A) Linear, (B) branched, (C) lightly cross-linked, and (D) highly cross-linked (Safranski & Griffis, 2017).

12.2 Various shape-memory polymers

accomplished very easily with the help of exposure of radiation like ultraviolet (UV) light, electron beam, gamma, etc. or heat (Hearon et al., 2013; Yakacki et al., 2008). There are two types of cross-linking: (1) chemical and (2) physical. When link between the molecules is created by covalent bonds, it is known as chemical cross-linking, for example, methacrylate or epoxies networks. On the other hand, when link between the molecules is generated by hydrogen bonding, phase separation, or physical entanglements, it is called physical cross-linking, for example, thermoplastic polyurethanes. Generally, cross-linking is measured by the cross-linking density. With the help of theory of elasticity of rubber, the cross-linking density is determined in the form of modulus by Eq. (12.1) (Pascault et al., 2002): ξ 5 3RTρ


where ξ is the elastic modulus, R is the universal gas constant, T is the temperature in Kelvin, and ρ is the cross-link density.

12.2.2 Thermal transitions The glass transition is kinetic phenomenon and considered a second-order phase transition—the temperature over which the amorphous polymer material comes into a flexible rubbery state from a brittle glassy state as they are heated. This temperature is known as glass transition temperature. The polymer structure keeps glassy state and it is rigid in nature with some small degree of molecular motion below the glass transition temperature. While the polymer structure becomes flexible, higher degree of molecular motion is achievable above the glass transition temperature. There are usually three techniques utilized to measure the glass transition: (1) differential scanning calorimetry (DSC), in which a step-change occurs in the heat capacity of the polymer during the glass transition; (2) dynamic mechanical analysis (DMA), in which a dramatic decrease in storage modulus signifies the onset of the glass transition or the peak of the tan delta is often used to represent the glass transition temperature, even though the glass transition occurs over a temperature range; and (3) thermomechanical analysis, in which a change in volume or a change in the coefficient of thermal expansion occurs when heating through the glass transition. The temporary shape can be locked in the SMPs with the help of cooling after glass transition, due to which modulus and viscosity increase rapidly. The glass transition temperature depends upon compositions and chemical structure of polymers; hence it varies broadly for a variety of polymers. For example, (meth)acrylates possess glass transition from 223 C to 112 C (Safranski & Gall, 2008). The shape-memory cycle can also use crystallization of polymer chains. After melting, cooling process is performed, and due to which some polymers begin to organize in the form of crystalline lamellae, they are folded upon themselves and known as stacked polymer chains. Further, these crystalline lamellae may arrange in the form of bigger crystalline spherulites. Moreover, mostly polymers possess crystalline and amorphous both regions; thus



CHAPTER 12 Shape-memory polymers

they come in the category of semicrystalline. Because when some sections of the polymer chains are folded and stacked in these lamellae, a section remains without stack in the amorphous region outside of the crystalline lamellae. Chain mobility is constrained during crystallization, and due to which a temporary shape becomes programable for generating SME. This is usually happened by a huge enhancement in modulus as the polymer cools after reaching glass transition (Nielsen & Landel, 1994). On the other hand, above glass transition the melting process takes place, due to which polymer spherulites and lamellae lose their arranged stacking and revert into nonarranged melt. This revert process of lamellae disorder permits the motion of polymer chains at huge scale resulting to bring original structure of SMPs. The complete features in terms of crystallization and melt transitions of SMPs can be seen by DSC. In the DSC scan, exothermic peak is associated with crystallization transition and endothermic peak is related to melt transition.

12.2.3 Categorization of shape-memory polymers Chemical structures of SMPs are responsible to categorize them with different properties. According to Mather (Liu, Qin, & Mather, 2007), SMPs can be divided into four core groups. Chemically cross-linked matrix that employs a glass transition for their activation is first classification. Chemically cross-linked matrix that employs a melting transition for their activation is second classification. Third classification is physically cross-linked polymer that employs a glass transition for their activation, while physically cross-linked polymer that employs a melting transition for their activation comes in the fourth classification.

12.3 Mechanism of shape-memory polymers The internal energy plays an important role in SMPs, because whole mechanism of SMPs depends upon internal energy. The mechanism can be assumed in the way, through analyzing an alteration in entropy, when strain energy is stored and released in the SMPs system (Lendlein & Kelch, 2002). In undeformed or original state, the SMPs possess very low entropy. When temperature of SMPs increase up to activation energy or above, they start to deform and attain higher entropy state. Here, strain energy can be locked on cooling. At this stage, SMPs achieve metastable state at high energy. Due to vanishing kinetic energy at cooling, the melt transition or glass transition inhibits SMPs to return at low or original energy state. Further, they gain activation energy on raising temperature above melt transition or glass transition. At this stage, available polymer chains come in moveable condition again by discharging stored strain energy resulted decrease in entropy and arrive back to their original state or shape. There are two significant practical aspects (1) cross-links and (2) switching segments of molecular structure

12.4 Composites using shape-memory polymers

of SMPs. With the help of cross-links, the SMPs are able to memorize their original specific shape, while reversible shape modification experiences by switching segments, which are the polymer chains existing between cross-links. In the switching segments, on increasing temperature above definite transition temperature, the polymer chains become extremely elastic. Hence, they can be deformed certainly and can produce huge strain at small stress. Hereafter, the polymer chains become immovable with securing temporary shape, when system is cooled beneath transition temperature. The one kind of transition is known as glass transition at which polymer chains become inelastic, because at this stage they leg behind the activation energy for basic movement. On the other hand, transition is recognized as melt transition at which system latches the impermanent shape by precluding the polymer chains from returning to the original perpetual shape. The schematic representation of mechanism for three types of polymers with two types of transition temperature and structures is illustrated in Fig. 12.3 (Lendlein & Kelch, 2002).

12.4 Composites using shape-memory polymers Polymer composites consist of polymer network with fillers, including various physical and chemical properties. The size and properties of fillers play a significant role in polymer composites, which can enhance the existing property of polymer composites. The functionalization of SMPs in terms of polymer composites with magnetic, optical, electrical, and biological fillers has been described in this section. The fillers can be in the particle, sheet, and tubes form.

FIGURE 12.3 Schematic representation of the molecular mechanism of the thermally induced shapememory effect for (A) a multiblock copolymer with Ttrans 5 Tm, (B) a covalently crosslinked polymer with Ttrans 5 Tm, (C) a covalently cross-linked polymer with Ttrans 5 Tg. If the increase in temperature is higher than Ttrans of the switching segments, these segments are flexible, and the polymer can be deformed elastically. The temporary shape is fixed by cooling down below Ttrans. If the polymer is heated up again, the permanent shape is recovered.



CHAPTER 12 Shape-memory polymers

12.4.1 Functionalization of shape-memory polymers by silicate To enhance mechanical properties of SMP nanocomposites (SMPNCs), the layered silicate has been obtained a very efficient filler (Lendlein, 2010). It has also been observed from the literature (Lendlein, 2010) that layered silicate-doped SMPNCs have been achieved high value of Young modulus and heat resistance, while lower value of inflammability and gas permeability with higher level of biodegradability. The aspect ratio can be increased from 10 to 1000 by developing layer thickness of the order of 1 nm in layered silicate. There are three different kinds of morphological feature in nanocomposites as (1) intercalated, (2) flocculated, and (3) exfoliated, designed by interaction between polymer network and filler as layered silicate. The intercalated nanocomposites were developed and characterized by introducing polymer chain into layered silicate in the form of replication distance of some nm as crystallographic manner. Whereas flocculated nanocomposites are generated because of hydroxylated edgeedge interactions, intercalated stacked silicate layers start to flocculate. Moreover, exfoliated nanocomposites are fashioned when each specific silicate layer arranges separately in the polymer network with regular distances, which entirely depends upon the relative clay composition. The aggregation of silicate in the polymer network may occur due to deficiency of interaction between hydrophobic polymer and hydrophilic silicate. However, this problem can be solved by surface modification of filler, which makes compatible interaction with polymer network resulted significant enhancement in mechanical properties. It has been found that the silicate of high aspect ratio provides large surface area to stay in contact and make bond with polymer network. This higher order of silicate and polymer interaction due to large aspect ratio of silicate generates greater barrier and mechanical properties than polymer network alone (Pinnavaia & Beall, 2000). This enhancement in mechanical properties could be accredited by elevated toughness with high strength, where silicate layers are randomly dispersed in polymer network, which propagate stress in matrix and impact strongly on the mobility of polymeric chain (Lietz et al., 2007; Yang, Zhang, Schlarb, & Friedrich, 2006). It is very well demonstrated that dispersion of silicate layers in polymer network reduces the mobility and creates short-range order in alignment of polymer chain. Hence, the development of resistance to drive polymer chain due to stress is increasing the toughness and modulus in composites (Yang et al., 2006).

12.4.2 Functionalization of shape-memory polymers by magnetic particles The functionalization of SMPs using magnetic particles can bring many features in composites with enhanced properties. Cobalt, nickel, iron, and their composites possess ferromagnetic or ferrimagnetic properties. These magnetic materials attract toward strong magnetic field and come back after removing field. Without magnetic field the magnetic domains arrange randomly, hence generate net

12.4 Composites using shape-memory polymers

magnetic moment zero. As magnetic field is applied, the magnetic domains start to arrange in the direction of magnetic field and generate strong magnetization within the domain region. In this process, ferromagnetic or ferrimagnetic particles can produce heat through eddy current and hysteresis loss in an alternating magnetic field, which depends upon different kinds and sizes of the magnetic particles (Goldman, 1990). The magnetic particles interact with the external magnetic field, SMP network, and with each other through Zeeman term, elastic deformation, and demagnetization field, respectively (Conti, Lenz, & Rumpf, 2007). When an alternating magnetic field is applied to SMP composite functionalized with magnetic particles, the temperature of the composite increases. As temperature reaches above the switching temperature of SMP network, the SMP has probability to recover its permanent or original shape. Whenever SMP could not trigger the SME via direct heating on increasing surrounding temperature, then this noncontact indirect heating technique via magnetization could be used to actuate SME. The size of the magnetic particles can alter the amount of produced heat via an alternating magnetic field because they are directly related to each other. The core size and concentration of magnetic particles can be measured using vibrating sample magnetometry (Kurchania, Rathore, & Pandey, 2015). At high magnetization frequency the nanoparticles have capability to generate enough heat needed for stimulated SME (Rosensweig, 2002). For controlling the amount of heat in the system, the applied frequency acts as a significant parameter. If the size of the magnetic particles is at the micrometer scale, then on applying magnetic field, the eddy current loss and hysteresis loss become higher and generate excess amount of heat in the low-frequency range. Consequently, agglomeration and higher size of the particles are not suggested for inducing SME specially in the medical field, because extra heating may cause serious damage of nearby tissues. Some specific ferromagnetic particles contain feature of thermoregulation because of Curie temperature (TC), which appears on producing heat by magnetic particles of suitable size in an alternating magnetic field. The ferromagnetic particles have capability to generate heat up to TC. Above TC ferromagnetic material alters in paramagnetic and cannot produce heat through the process of hysteresis loss. Because ferromagnetic material with appropriate TC acts like a thermostat, hence it could substantially decrease the risk of excess heating in biomedical applications. Below TC ferromagnetic nanomaterials (below 15 nm, single domain) possess superparamagnetic behavior, in which the energy needed to alter the direction of magnetic moments present in single domain is analogous to the surrounding thermal energy. The magnetic moments will start to reverse randomly at a significant rate. In the absence of applied magnetic field, superparamagnetic materials do not hold any substantial magnetization as ferromagnetic materials possess, hence they do not accumulate. The applied magnetic field orients all the magnetic moments in superparamagnetic (single domain) material in its own direction. This orientation enhances the strength of applied magnetic field in its vicinity. On removing magnetic field, Brownian motion begins to mix up magnetic moments resulted material’s demagnetization.



CHAPTER 12 Shape-memory polymers

12.4.3 Functionalization of shape-memory polymers by carbon fillers The functionalization of SMPs with carbon compounds has brought in knowledge where they use as fillers [e.g., carbon fibers (CFs), carbon black, carbon nanorods (CNRs), graphite, etc.]. All these carbon fillers are extremely conductive and reduce electric resistance of composites significantly. Hence, conductive SMPs can be produced in terms of SMPC. These SMPC has capability to trigger through small amount of heat by indirect actuation process. Due to the abovementioned carbon fillers, electricity can be conducted in the plane of all covalently attached layers to create electrons-cloud because of delocalization of outer electrons. Thus the smaller bulk electrical conductivity for carbon generated than for metals. Although metals in both nano and bulk forms are more conductive than any kind of carbon fillers, still carbon fillers have some atmospheric quality. Metals have tendency to corrode and form a nonconductive layer on their surface due to oxidation process while carbon forms two-dimensional flat graphite sheet at atmospheric pressure, in which carbon atoms are weakly bounded in the form of hexagonal rings by van der Waals forces resulting graphite has cleaving properties with softness. The layers slip very easily on another in the graphite. Hence these layers can be folded cylindrically in the form of carbon nanotubes (CNTs) either in single-walled (SWCNT) or in multiwalled (MWCNT) (Harris, 2009). Carbon nanofibers (CNFs) are also easily available in huge quantities at a suitable price. Due to very large diameter B100 nm, CNFs are different from CNTs (SWCNTB1-nm diameter and MWCNTB10-nm diameter). On the other hand, CFs are thin fibers of approximately 5- to 10-μm diameter. In CFs, carbon atoms are microscopically bonded as crystals and aligned parallel along the axis of fiber. This kind of crystalline arrangement of carbon atoms makes the fibers usually stronger in the same dimension. The functionalization of SMPs using carbon compounds brings new opportunities in the field of composite. The polymer composites using SMPs as polymer matrix and CNTs as fillers have been prepared by many researchers in the last decade. It has been observed from literature (Lendlein, 2010) that the presence of CNTs in small concentration can enhance the mechanical and thermal properties, which can also generate exceptional electrical properties with improvement in the behavior of SMPs. Due to anisotropic behavior of CNTs, they show percolative nature at small concentration as they use like fillers in the SMPs network. The diameter in the nano range and excellent electrical and mechanical properties of CNTs offer an exceptional opportunity to enhance the structural strengthening and thermal controlling of a polymer network. The functionalized SMPs with CNTs are conducting composites in terms of SMPCs and can generate SME’s actuation on applying electric field rather than by providing heat through atmospheric temperature. Hence, for controlling microaerial vehicles, SMPCs are being employed as electroactive actuators (Paik, Goo, Jung, & Cho, 2006). Some chemical properties like interfacial adhesion, homogenous distribution as well as

12.4 Composites using shape-memory polymers

compatibility of CNTs with polymer matrix have been accomplished by surface modification using mixture of sulfuric and nitric acids (Lin et al., 2003). Usually, insertion of carbon compounds as filler in the matrix of SMPs may not only considerably modify the polymeric behavior as thermoplastic elastomers but also enhance several characteristic features of composites.

12.4.4 Functionalization of shape-memory polymers by biocompatible materials The biocompatible material hydroxyapatite or hydroxylapatite (HA— Ca10(PO4)6(OH)2) is a mineral appearing in nature in the crystalline hexagonal form, in which crystal unit cell contains two molecules of Ca5(PO4)3(OH). Around 70% of inorganic HA mineral is being comprised by natural bone and teeth. Hence, HA is very vital bioactive material which uses in orthopedic, dental, and maxillofacial applications, it supports bone ingrowth and osseointegration (Jarcho, 1981). Hence, it is used as a filler while replacing bone and as a coating to promote bone ingrowth into prosthetic implants. The mechanical properties and protein adsorption capacity of the SMPCs experience great improvement when nano-HA are introduced to it (Labella, Braden, & Deb, 1994). It has been observed that proteins in mineralized tissues acted as nature’s crystal engineers, playing a pivotal role in promoting or inhibiting the growth of minerals such as hydroxyapatite. It is to be noted that pure β-tricalcium phosphate (β-TCP), Ca3(PO4)2, does not exist in nature or in any biological system and cannot be prepared by direct precipitation and hydrolysis methods. It can be synthesized by heating calcium-deficient apatite of appropriate Ca/P molar ratio above 800 C or by heating amorphous calcium phosphate. In terms of solubility and in vivo biodegradation, both HA and β-TCP were found to be different. But displayed osteoconductive properties. A carrier matrix for bioactive agents was provided by porous β-TCP material and it formed a moldable putty composition when a binder was added. The use of HA and β-TCP in wide applications in hard tissue implantations has been limited owing to their poor mechanical properties such as low strength and fracture toughness (De Groot, De Putter, Smitt, & Driessen, 1981). But composites from biocompatible SMPs and HA or β-TCP have shown improved mechanical properties resulting in the introduction of these composites in wide-ranging medical applications. The nanocomposites of hydroxyapatite (HA-Ca10(PO4)6(OH)2), poly(DL-lactide) (PDLL)/β-TCP have proved to be an outstanding biomaterial used in tissue engineering and have also been used clinically in various forms (Zheng et al., 2008). The preparation of these nanocomposites with different β-TCP is same as the preparation of PDLL/HA nanocomposites described in literature (Lendlein, 2010). As detected by laser diffraction particle size analyzer, average particle size of β-TCP used in this work was approximately 720 nm with particle distribution of 2001500 nm. Phosphate buffer saline solution at 37 C (PBS, pH 5 7.4) was used to investigate the



CHAPTER 12 Shape-memory polymers

hydrolytic degradation process while studying the effect of in vitro degradation on the shape-memory capability of PDLL/β-TCP nanocomposites. The specimens were removed from degradation medium and rinsed with distilled water to remove residual buffer salts at fixed intervals and then put in vacuum for drying. The SME of PDLLA/β-TCP and pure PDLLA composites with different β-TCP were examined after and before dipping in the buffer solution. Bending test was conducted for studying the shape recovery. Noticeable differences were observed in the behavior of their shape memory at different intervals of degradation time. The dependence of shape recovery ratio (RR) on degradation time as calculated according to given Eq. (12.2) (Lendlein, 2010), RR 5

180 2 final angle 3 100 180


The SMPCs were folded by 180 for programming. RR decreases when in vitro degradation time in PBS increases. Keeping degradation time constant, the RR for PDLLA/β-TCP composites was way more than that of pure PDLLA. RR was minutely higher for all composites at the 21st day of degradation time as reported (Lendlein, 2010). This may be due to breaking of PDLLA chains or crystal phase changes of β-TCP particles. The following reaction formula depicts how in vitro degradation of PDLLA/β-TCP composites can result in Ca2P2O7, CaHPO4, and HA phases (Yakacki et al., 2008): 4β 2 Ca3 ðPO4 Þ2 1 H2 O-Ca2 P2 O7 1 Ca10 ðPO4 Þ6 ðOHÞ2 ; Ca2 P2 O7 1 H2 O-2CaHPO4


Due to a plasticizer effect, reduction of Tg can take place when new inorganic phases are formed (Yang, Huang, Li, & Chor, 2005). The existence of Ca2P2O7, CaHPO4, and HA particles aids in the shape recovery ratios after 21 days of degradation time due to imparting more constraints on polymer chain dynamically (Prokop, Jubel, & Hahn, 2005). Shape memory may become undesirable as the degradation time increases. This may be due to PDLLA chains breaking and dissolution degradation of the inorganic phases in PDLLA/β-TCP composites.

12.5 Limitations of shape-memory polymers There are two fundamental limitations of SMPs. First one describes about recovery time and activation process, while second discusses about recovery force and work capacity in the following section.

12.5.1 Recovery time and activation process The recovery time of SMPs depends on difference between their activation temperature and the operating temperature. If activation temperature is very near to

12.5 Limitations of shape-memory polymers

their operating temperature, system takes only some minutes in recovery, while activation temperature is very far above from the operating temperature, system may take long time as hours or days to recover its previous shape. According to Yakacki et al. (2008), the SMPs have capability to recover within a couple of minutes if the operating temperature is nearby to the activation temperature, which is a glass transition of given material as illustrated in Fig. 12.4. Hence, for fast recovery, quick heat transfer is required because most polymers are thermal insulators. Usually, in various applications, heating is carried out in the presence of air. Hence, for rapid recovery a wet atmosphere is suitable due to maximum heat transmission (Lakhera et al., 2012a). According to preferred application, activation process has many advantages and drawbacks. The thermal activation can be comparatively improved by providing heat using energy or heat source. Thus big problem may occur as quick thermal activation, if there is no control on heat source. Here, thermal processes are attaining more emphasis rather than classic thermal processes for activation. To recover previous shape a solvent-induced activation may perform, particularly in wet biological atmospheres where utilizing a heat source is not possible. In this case, recovery of SMPs completely depends on the consuming rate and quantity of water. Consequently, it may take some minutes to hours for recovery, which may be adequate for preferred application. These activation techniques are not like mechanically sponsored activation, which basically uses mechanical energy to apply force on SMPs to bring them at low energy level from its high energy level (Safranski & Griffis, 2017; Yakacki, 2008). This mechanical activation can be performed quickly without any use of

FIGURE 12.4 Strain recovery profile (%) at operating temperature (Top 5 50 C) for three different SMP networks (1, 2, and 3) as function of time (min) with varying transition temperature (Ttrans) (Yakacki et al., 2008). SMPs, Shape-memory polymers.



CHAPTER 12 Shape-memory polymers

solvent or thermal energy. Nevertheless, SMPs must have capability to bear the distortion and applied forces without rupture during returning back to their previous shape.

12.5.2 Recovery force and work capacity As a consequence of forced or partially forced recovery, SMPs act as actuators driven by the stored energy from programming. Many parameters on which stored energy is dependent include temperature, strain and deformation rate, the programming conditions that determine the deformation capacity of SMPs. There is a trade-off in the failure strain as a function of programming temperature for many SMPs that are chemically cross-linked and have a glass transition (acrylics, epoxies, some polyurethanes). Hence there occurs an optimal temperature near the onset of the glass transition (Safranski & Gall, 2008) that exists for programming as shown in Fig. 12.5. The SMP should be programmed at this optimal temperature to achieve the largest recovery forces, so that most of the energy is put into the polymer during deformation (e.g., a high strength and high strain actuator). As examined by Lakhera (Lakhera et al., 2012b), mechanical work can also be performed by SMPs. It was found that under a constant amount of

FIGURE 12.5 Failure strain as a function of temperature relative to glass transition temperature for two networks at 2.5 mol% PEGDMA550. Applied for permission from Elsevier, Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and cross-linking density on the thermo-mechanical properties and toughness of (meth)acrylate shape memory polymer networks. Polymer, 49(20), 444655.

12.5 Limitations of shape-memory polymers

programming strain, mechanical work improves with cross-linking density (i.e., rubbery modulus). Under partially constrained conditions, when a bias force is applied until the fully constrained condition is reached, the recovery strain decreases in a linear manner. As the bias force decreases the recovery strain to 50% of its unconstrained value, the mechanical work displays a maximum. This factor can be of great help in the designing best of SMP actuators just by knowing the maximum amount of possible work that can be done and the distance the actuator will move during deployment. It is to be noted that these results were observed at a constant programming strain for polymers with varying crosslinking densities (i.e., different spring stiffness constants) and under compression conditions. Stress and strain change as a temperature change for SMPs, although work is a function of stress and strain. This state is usually employed to establish the recovery stress, σr , which can be applied by the SMPs. This is known as permanent-strain recovery as the quantity of strain is set up to a constant value, which can be seen by Eq. (12.4) (Safranski & Griffis, 2017), σr  Er Amax


where Er is the rubbery modulus and Amax is the maximum programming strain. For a given SMP, work can be maximized when programming occurs at its

Table 12.1 Thermomechanical properties of networks composed of 90 mol% tBA and 10 mol% multifunctional (meth)acrylate. Multifunctional (meth)acrylate

Tg ( C)

Er (MPa)


22.75 70.5 43.5 64.5 59.5 62.5 68.5 40.5 98 83 55 24.5 58 69.5 92 74

7.35 8.15 8.25 9.0 8.8 6.48 10.85 10.7 42.5 25 16.65 15.95 23 15.5 49.5 129.5

Source: Applied for permission from Elsevier, Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and cross-linking density on the thermo-mechanical properties and toughness of (meth) acrylate shape memory polymer networks. Polymer, 49(20), 444655.



CHAPTER 12 Shape-memory polymers

optimal temperature and programming strain is increased. The thermomechanical properties of networks composed of 90 mol% tBA and 10 mol% multifunctional (meth)acrylate are tabulated in Table 12.1.

12.6 Conclusion The SMPs have been discussed as actively moving materials, which come in the category of smart materials. SMPs have been defined by three steps: programming, storage, and recovery, in which SMPs alter their shape at ambient temperature, lock it on cooling, and further regain it. The molecular mechanism of the thermally induced SME for different polymers has been illustrated precisely. The functionalization of SMPs with silane, carbon nanomaterials, and magnetic nanomaterials prepares them for various applications. The biocompatible SMPs are being used in orthopedic, dental, and maxillofacial applications, and it supports bone ingrowth and osseointegration. The limitations of regain their shape have been described by strain recovery profile as function of time with varying transition temperature and failure strain as a function of temperature relative to glass transition temperature of different SMP networks.

References Conti, S., Lenz, M., & Rumpf, M. (2007). Journal of the Mechanics and Physics of Solids, 55, 1462. De Groot, K., De Putter, C., Smitt, P., & Driessen, A. (1981). Journal of Ceramic Science and Technology, 1, 433. Goldman, A. (1990). Modern ferrite technology. New York: Van Nostrand Reinhold. Harris, P. J. F. (2009). Carbon nanotube science: Synthesis, properties and applications. New York: Cambridge University Press. Hearon, K., et al. (2013). Electron beam crosslinked polyurethane shape memory polymers with tunable mechanical properties. Macromolecular Chemistry and Physics, 214, 12581272. Jarcho, M. (1981). Clinical orthopaedics, 157, 259. Kurchania, R., Rathore, D., & Pandey, R. K. (2015). Studies on size dependent strain and nanomagnetism in CoFe2O4 nanoparticles. Journal of Materials Science: Materials in Electronics, 26, 93559365. Labella, R., Braden, M., & Deb, S. (1994). Biomaterials, 15, 1197. Lakhera, N., et al. (2012a). Biodegradable thermoset shape-memory polymer developed from poly(β-amino ester) networks. Journal of Polymer Science, Part B: Polymer Physics, 50, 777789. Lakhera, N., et al. (2012b). Partially constrained recovery of (meth)acrylate shape-memory polymer networks. Journal of Applied Polymer Science, 126, 7282. Lendlein, A (Ed.), (2010). Advances in Polymer Sciences: Shape Memory Polymer. Springer.


Lendlein, A., & Kelch, S. (2002). Shape-memory polymers. Angewandte Chemie— International Edition, 41, 20342057. Lietz, S., Yang, J. L., Bosch, E., Sandler, J. K. W., Zhang, Z., & Altsta, V. (2007). Improvement of the mechanical properties and creep resistance of SBS block copolymers by nanoclay fillers. Macromolecular Materials and Engineering, 292, 23. Lin, Y., Zhou, B., Fernando, K. A. S., Liu, P., Allard, L. F., & Sun, Y. P. (2003). Polymeric carbon nanocomposites from carbon nanotubes functionalized with matrix polymer. Macromolecules, 36, 7199. Liu, C., Qin, H., & Mather, P. T. (2007). Review of progress in shape-memory polymers. Journal of Materials Chemistry, 17, 15431558. Meng, H., & Li, G. (2013). A review of stimuli-responsive shape memory polymer composites. Polymer, 54, 21992221. Nielsen, L. E., & Landel, R. F. (1994). Mechanical properties of polymers and composites (2nd (ed.)). New York: Marcel Dekker. Paik, H., Goo, N. S., Jung, Y. C., & Cho, J. W. (2006). Development and application of conducting shape memory polyurethane actuators. Smart Materials and Structures, 15, 1476. Pascault, J. P., et al. (2002). Thermosetting polymers. New York: Marcel Dekker. Pinnavaia, T. J., & Beall, G. W. (2000). Polymer-clay nanocomposites. New York: Wiley. Prokop, A., Jubel, A., & Hahn, U. (2005). Biomaterials, 26, 4129. Rosensweig, R. E. (2002). Heating magnetic fluid with alternating magnetic field. Journal of Magnetism and Magnetic Materials, 252, 370. Safranski, D. L., & Gall, K. (2008). Effect of chemical structure and crosslinking density on the thermo-mechanical properties and toughness of (meth)acrylate shape memory polymer networks. Polymer, 49, 44464455. Safranski, D. L., & Griffis, J. C. (2017). Shape-memory polymer device design. Atlanta, GA, United States: MedShape, Inc. Yakacki, C. M., et al. (2008). Strong, tailored, biocompatible shape-memory polymer networks. Advanced Functional Materials, 18, 24282435. Yang, B., Huang, W. M., Li, C., & Chor, J. H. (2005). Effects of moisture on the glass transition temperature of polyurethane shape memory polymer filled with nano-carbon powder. European Polymer Journal, 41, 1123. Yang, J. L., Zhang, Z., Schlarb, A. K., & Friedrich, K. (2006). On the characterization of tensile creep resistance of polyamide 66 nanocomposites. Part II: Modeling and prediction of long-term performance. Polymer, 47, 2791. Zheng, X., Zhou, S., Yu, X., Li, X., Feng, B., Qu, S., & Weng, J. (2008). Effect of in vitro degradation of poly(D,L-lactide)/β-tricalcium composite on its shape-memory properties. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 86B, 170.



Rapid prototyping


Umesh K. Dwivedi1, Shashank Mishra2 and Vishal Parashar2 1

Amity School of Applied Sciences, Amity University Jaipur, Jaipur, Rajasthan, India Department of Mechanical Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India


13.1 Introduction Rapid prototyping (RP) is an interdisciplinary approach in the field of manufacturing industry that has a potential to bring a revolutionary change in prototyping. The methodology employed in the design and development of parts using RP systems is novel and dissimilar from the conventional process. RP as evident by its name is a technology capable of quick fabrication of prototypes. It was developed by Charles Hull in 1986, since then the technology has become a thrust field of attention for researchers and design engineers. The first RP technology was based on the SLA process and termed as stereolithography. Because of advantages like flexibility and ease of customization over other conventional processes, these systems attract the attention of the researchers. Expiry of the patents associated with these systems opened an opportunity for progress and the technology evolved rapidly after that. There are several examples of industrial applications employing RP systems for production, for example, a group in China has developed cheap houses causing the cost of the house as low as $4000. A wide variety of materials are used in these systems for the development of parts that employ thermoplastics like polylactic acid (PLA), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), and nylon. Some costly RP processes are utilizing metal alloy powders as feed materials. Ceramics are used for the development of three-dimensional (3D) printed scaffolds. Depending upon the method these systems take up raw materials in different physical forms like powder, liquid, slurry, and filaments. A prominent application associated with 3D printing systems includes the development of custom orthoses and prostheses. According to Chang et al. micro-manufacturing (MEMS, microelectromechanical systems) is also being done using RP processes (Blok, Longana, Yu, & Woods, 2018; Chang, 2015; Chen, Jin, Wensman, & Shih, 2016; Chua, Leong, & An, 2020; Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016; Ngo, Kashani, Imbalzano, Nguyen, & Hui, 2018; Wang, Jiang, Zhou, Gou, & Hui, 2017). Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 13 Rapid prototyping

In RP, the computer-drafted designs are fabricated layer by layer and bonding them together using automatic and smart manufacturing processes; thus the final product is apparently printed, because of which this method is also termed as additive manufacturing or 3D printing. Along with mass customization, these printers do not employ any tool for part machining. Also, there is absolutely no wastage of material in the fabrication processes. On contrary, most of the conventional machining processes use the subtractive approach for fabrication that results in material wastage. MacDonald and Wicker (2016) have mentioned that, with the progress in the commercial application of RP, these systems are now considered more than just prototyping technique. Because of the mass-scale production from this technology users are reluctant to use the term prototyping, that is why it is frequently termed as rapid manufacturing or additive manufacturing (MacDonald & Wicker, 2016). While most of the authors across the world use RP as a synonym for additive manufacturing, Gurr and Mu¨lhaupt (2012) have considered computer numeric controlled (CNC) machining as one of the types of RP as shown in Fig. 13.1. CNC machining operations are rapid in production and prototyping, but the fabrication methodology is subtractive in nature leading to a contradiction to call it as one of the additive manufacturing methods. Table 13.1 elaborates about the available 3D printing technology, their year of market entry, materials employed by these systems, maximum size of the parts, and the cost at which they are purchased.

FIGURE 13.1 Classification of rapid prototyping processes according to the initial state of the processed material and the principle of layer solidification (Gurr & Mu¨lhaupt, 2012).

Table 13.1 Key characteristics for comparison (Kumbhar & Mulay, 2018). RP process SLA FDM SLS 3DP 3D bioplotting LOM

Materials Photocurable resins (acrylics and epoxies) Thermoplastics (ABS, PC) Metals and thermoplastics (PA12, PC) Thermoplastics, ceramics, metals Thermoplastics, hydrogels, ceramics Paper, polymer, metal, ceramic

Market entry

Max. part size (mm)

Dim accuracy

Cost/machine (h)



1500 3 600 3 500





914 3 610 3 914




700 3 380 3 580

,0.05 0.1


1998 2001

4000 3 2000 3 1000 150 3 150 3 140

0.0.1 0.0.1

.20.000 .150.000


550 3 800 3 500



Low/ medium Medium/ high Low Low/ medium Low/ medium

ABS, Acrylonitrile butadiene styrene; LOM, laminated object manufacturing; PC, polycarbonate; RP, rapid prototyping; SLA, stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing.


CHAPTER 13 Rapid prototyping

Since the last two decades, 3D printers are finding a lot of applications in biomedical and artificial implants. Some of the most crucial applications include surgical planning by the development of the replica of the organs. The printing of artificial soft tissues is one of the most significant biomedical applications achieved with the help of RP technology; others include development of implants, prosthetics, etc. (MacDonald & Wicker, 2016). Design and fabrication of multifunctional products by use of 3D printing have been achieved recently. MacDonald and Wicker (2016) have reported different types of such developments. Examples of such fabrication include the development of sensor, actuators, antennas etc. which is further discussed in the forthcoming sections. Such systems are capable of producing parts from hybrid materials. Giachini et al. (2020) have employed 3D printing technology to produce functionally graded material (FGM) with uniform stiffness gradient by employing two different materials and varying the composition of the two materials throughout the fabricated part (Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016). Fig. 13.2 shows the commercialization status of RP systems. Another noteworthy innovation in the field of RP is the design and development of fiber-reinforced composite (FRC) (Zindani & Kumar, 2019). There are several examples of manufacturing composite materials by employing rapid manufacturing processes. Matsuzaki et al. (2016) have produced jute fiberreinforced unidirectional thermoplastic composite using 3D printing technology. Composites are widely used in different applications including aircraft, automobile, biomedicals, etc. Fabrication of FRC employing additive manufacturing is the milestone in the sector of manufacturing. In the recent studies, it has been observed that the influential parameters in FRC like fiber geometry, stacking

FIGURE 13.2 Commercial relevance of rapid prototyping technologies. Revenues made concerning (A) industry and (B) field of application (Gurr & Mu¨lhaupt, 2012).

13.2 Preprocessing, the process, and postprocessing

sequence, etc. can also be altered using 3D printing. Natural FRCs employing RP systems are also at embryological stage (Krishna, Kate, Satyavolu, & Singh, 2019). In this chapter, various types of RP systems are explained; an attempt has been made to brief about the working principle and discuss all the variants in chronological order, till the latest development. Also, the associated terminology in relation to the additive manufacturing systems is defined to enhance the grasp on the content. In addition to that, the contemporary research and developments are detailed in the forthcoming sections to explore the future orientation and possibilities associated with these processes. Finally, the applications of additive manufacturing systems in vital sectors like biomedical and biomechanical engineering are also reported herein.

13.2 Preprocessing, the process, and postprocessing in rapid prototyping The fabrication methodology involved in RP undergoes the following three stages: • • •

Preprocessing The process Postprocessing

13.2.1 Preprocessing In this phase of the development, the part is usually drafted in the computerbased drafting software and the dimensioning is done. However, there are software that can produce a 3D draft directly by the images obtained after scanning the object at various orientations. This draft is then directly used for the next stage of development. Such types of methods are most suited for the development of an implant that can be used during surgical procedures. Scanning saves a lot of time for complex geometries that require intricate detailing, because of which this method finds frequent application in the field of medical science for the printing of implants (Kumbhar & Mulay, 2018). For printing a prototype, the images that are either drafted manually or developed by scanning any body organ are converted in Standard Tessellation Language (STL) format. STL file contains the draft in the form of small triangular facets, which are connected with each other producing the 3D image of the implant to be fabricated. The conversion of the computer-drafted Computer Aided Drafting (CAD) file to STL extension reduces the accuracy in the dimensions of the part to be fabricated. This compromise in the accuracy is a persistent shortcoming of 3D printing systems. The coordinates of the vertices of triangular facets and the direction of the outward normal are present within the STL file.



CHAPTER 13 Rapid prototyping

Because of the facets being triangular in shape, the curved surfaces not easily obtained, to achieve the desired accuracy the number of facets needs to be increased, that results in increase in file size. It is the responsibility of the designing engineer to balance the accuracy and file size as per the application of the implant being manufactured. Usually, the STL model is used as a universal format; however, another model that works on a similar principle is also available, known as stereolithography contour (Chang, 2015). Conversion of the drafted file in STL format is followed by slicing of the STL file which is to be developed using a RR system. The slicing is achieved with slicing software. Depending upon the type of 3D printer system employed for manufacturing the product the thickness of the slices vary from 0.0254 to 0.254 mm (Chang, 2015).

13.2.2 The process During this phase, the STL file is sliced in layers within the aforementioned thickness range using the slicing software. The layer thickness is a significant parameter of the product quality. The choice of layer thickness depends upon the purpose of the part/implant being fabricated as well as the type of 3D printing system being used. In the next step, platform within the printer also termed as the printing bed starts fabricating the sliced layers successively. Workflow can be further subdivided into three steps; first one is the supply of raw material for fabrication, followed by deposition material on a predefined contour for the development of layer mostly by application of heat, laser beam, or ultraviolet (UV). The final step is the upward or downward motion of the bed for the development of next layer. These steps are repeated until the part is fully fabricated. In the case of computer-aided manufacturing, the fabrication is assisted with programmable sensors, actuators, and robots for the required operation. The placement of workpiece is manual, and the operation codes are typed. The movement of tool to the workpiece and the machining operation is encoded through the programming. This is achieved with the help of motors, servo motors, microprocessors, and microcontrollers. The machining process is accompanied with the continuous flow of coolant. The part build is achieved by machining operations like turning, milling, drilling, etc. and hence, the manufacturing process is subtractive (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012).

13.2.3 Postprocessing The fabricated part is allowed to cool/cure until it attains room temperature. Consecutively, the surface treatment is done and the method of surface treatment depends on the type of system employed for the fabrication. Fig. 13.3 shows the fabrication steps in 3D printing and conventional manufacturing processes. It is intended to perform a comparison among the former and the later. Fig. 13.3A shows the development and fabrication of product employing additive

13.3 Contemporary rapid prototyping systems

FIGURE 13.3 Diagrammatic comparison of different manufacturing processes: (A) 3D printing, (B) milling, and (C) molding (Gurr & Mu¨lhaupt, 2012).

manufacturing techniques. Fig. 13.3B is a subtractive manufacturing method involving machining operation like milling. Fig. 13.3C shows the casting process, that is one of the most orthodox methods of fabrication and manufacturing. With the advancements in additive manufacturing systems, it can be observed that the researchers have started focusing on the improvement in the quality of the product like the surface finish of the fabricated part, the dimensional accuracy, mechanical properties, etc. The improvement is achieved by the identification of the parameters that influence the quality of the end product. Depending upon the type of 3D printer, these parameters can be different, for example, printing speed in SLA, heat supplied, and nozzle diameter in FDM, voltage in laser engineered net shaping (LENS), etc. These parameters are then altered one by one to check the degree of dependency on individual parameters and figure out the optimal conditions for best results. An illustrative example of these parameters is shown in Fig. 13.4 (Blok et al., 2018).

13.3 Contemporary rapid prototyping systems According to the American society of testing and manufacturing (ASTM) F42 standard provided in the year 2009, there is a total of seven different types of



CHAPTER 13 Rapid prototyping

FIGURE 13.4 Main parameters for good surface contact and temperature conditions to enable optimal polymer sintering conditions (Blok et al., 2018).

systems that can be considered under the category of RR. This standard defines the terminology, methods for testing, and provides the standard file formats along with other relevant information and concepts. The list of various RR systems is available below: 1. 2. 3. 4. 5. 6. 7.

VAT polymerization Powder bed fusion Binder jetting Direct energy deposition Sheet lamination Material extrusion Material jetting

More elaborate data about the types of RP systems available in the market are provided in Table 13.2. In the scope of this chapter, all the systems as defined in ASTM F42 standard have been detailed to enhance the understanding of the contemporary additive manufacturing technology. Systems have been classified based on the form of the raw material supplied as the input in the printer as shown in Fig. 13.5 (Kumbhar & Mulay, 2018). The classification of RP systems has been reported on the basis of materials used. Table 13.3 shows the aforementioned classification. The physical form in which these materials are fed depends on the type of 3D printing system. Table 13.4 lists the different types of systems that require their feedstock in the form of solid, powder, and liquid, respectively.

13.3 Contemporary rapid prototyping systems

Table 13.2 Examples of various AM processes on basis of raw material input (Kumbhar & Mulay, 2018). Working principle

Manufacturing process

VAT photopolymerization Material extrusion Material jetting Binder jetting Sheet lamination Powder bed fusion

Stereolithography FDM Drop-on-demand Binder jet LOM Direct metal laser sintering Electron beam melting (EBM) Selective heat sintering SLS 3DP LENS, DMD

Directed energy deposition

LENS, Laser engineered net shaping; DMD, direct metal deposition, LOM, laminated object manufacturing; SLS, selective laser sintering; 3DP, three-dimensional printing.

3D Printing

Solid Raw Material

Powder Based


Laminate Based


Liquid Raw Material

Filament Based

VAT Polymerisation




FIGURE 13.5 Classification of the available additive manufacturing processes.

13.3.1 Available rapid prototyping systems The systems that fall within the domain of the ASTM F42 and have standards for research and experimentation are available directly from the aforementioned manual. At least one system from all the seven variants has been described in the forthcoming pages of this chapter. Fig. 13.5 shows classification of different types of 3D printing systems on the basis of physical form of the feedstock for the seven different variants mentioned in ASTM F42.



CHAPTER 13 Rapid prototyping

Table 13.3 Classification based on the type of material used (Kumbhar & Mulay, 2018). Feed material

Employed Additive Manufacturing process


Three-dimensional printing (3DP) Selective laser sintering (SLS) FDM 3DP SLS Direct metal laser sintering Stereolithography Fused deposition modeling (FDM) 3DP SLS Laminated object manufacturing



Table 13.4 Rapid prototyping process based on form of raw material (Kumbhar & Mulay, 2018). Supply phase

Additive manufacturing processes






Materials Polymers (ABS, polyacrylate, etc.)wax, metals and ceramics with binder Ceramic, polymer, and metal powder with binder Photopolymer(epoxies, acrylate, filled resin, colorable resin)

ABS, Acrylonitrile butadiene styrene; DMLS, direct metal laser sintering; LOM, laminated object manufacturing; SLA, stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing. Selective laser sintering Selective laser sintering (SLS) was developed in 1986 at the University of Texas by Deckard. This is a powder-based system that requires polymer material in powder form, because of which it is also termed as powder bed system. Laser beam is applied for heating and melting of the powder, on specified contour. The powder then melts and bonds to produce the first layer of the powder bed, and then descends by a magnitude of one layer thickness in the downward direction. The layer thickness of the SLS printing process thus is an influential parameter in the product quality. After the bed descends, a fresh layer of powder is rolled over the bed. The process repeats until the part is developed (Kumbhar & Mulay, 2018; Tiwari, Pande, Agrawal, & Bobade, 2015; Chitresh, Singh, & Himanshu, 2014). Gurr and Mu¨lhaupt have presented a detailed review of various kinds of

13.3 Contemporary rapid prototyping systems

material used in the process of selective laser sintering which include thermoplastic polymers like high density polyethelene (HDPE), poly ether-ether Ketone (PEEK), etc. and composites that include fillers like glass beads, nano-silica, carbon nanofiber, nano-alumina, etc. These systems are also capable of direct sintering of ceramics and metals, like zirconia, alumina, silicon carbide, and stainless steel, titanium, bronze, and nickel, respectively (Gurr & Mu¨lhaupt, 2012). Selective laser melting The process of selective laser melting (SLM) is similar to the previous process, that is SLS but it is more efficient and frequently used for the part developed using metallic powder or sometimes a mixture of more than one metallic powder. In the process of SLM the parts fabricated exhibit properties almost similar to the conventional machining process. However, the cost of producing metal powder is high but on the other side, the intricate and complex geometry produced from RP technology can be considered as compensation to the aforementioned hard work of producing metal powder (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012; Kumbhar & Mulay, 2018). Chang (2015) has covered a detailed review about the types of processes involved in producing parts from metal, which include the technologies that use additive manufacturing from powdered metal, like LENS and electron beam melting (EBM). EBM employs titanium powder to fabricate parts; titanium is a material that is frequently employed in biomedical implants because of its biocompatibility, noncorrosive and nontoxic behavior. Fig. 13.6A is the diagrammatic representation of EBM technology, and the second part of the same figure, that is (B), is an image

FIGURE 13.6 The EBM technology of Arcam: (A) electron beam melting diagram. (B) Typical Arcam A2 system capable of producing parts with dimensions up to 7.87 in. 3 7.87 in. 3 13.0 in. (200 mm 3 200 mm 3 330 mm) (Chang, 2015).



CHAPTER 13 Rapid prototyping

FIGURE 13.7 The laser engineered net shaping (LENS) technology: (A) the LENS, (B) LENS fabrication, and (C) the Optomec LENS 850 system (Chang, 2015).

of the Arcam A2 system, an EBM 3D printer capable of producing parts of size of 200 mm 3 200 mm 3 330 mm in dimension. Fig. 13.7 shows a typical setup of the laser engineered net shaping also known as LENS technology. An actual physical setup of the system is shown in the third part of Fig. 13.7. Optomec’s RP machine works on the principle of LENS technology. The size of printing envelop of Optomec LENS 850 system is (18 3 18 3 42) in., with X and Y accuracy/resolution up to a level of 0.002 and 0.020 in. in the direction of the Z-axis. The LENS technology has a nozzle that push out the metallic powder and is melted simultaneously with the help of laser and thus the fabrication proceeds. However, this system does not require a powder bed kind of arrangement in contrast to its other substitutes (Chang, 2015). The challenge associated with LENS is the limitation of part size. These are good for small parts but very large metallic parts are difficult to be produced from these systems. There is a need of large setups, capable of manufacturing large metallic parts. On the positive side, these setups are capable in producing parts of size big enough to serve the requirement for biomedical implants. So, it can be successfully used for bioengineering-based applications. The other associated challenges with these setups include the metal shrinkage issue and the estimation of the allowance for the same. Also, the part printing takes a significant amount of time which produces a temperature gradient in the direction of printing. Under the influence of this temperature gradient the overall part develops a tendency to get distorted as the cooling proceeds with time. The distortion allowance is again a complex situation to deal with. Apart from this, warping is also observed in the fabricated part after they are cooled. It is also difficult to predict the grain size that will be produced in the part after it cools down. The significant parameter which affects the part quality includes the power associated with the laser, the melting point of the metallic powder, cooling time, nozzle diameter, etc. (Blok et al., 2018; Chang, 2015).

13.3 Contemporary rapid prototyping systems Laminated object manufacturing In the process of laminated object manufacturing (LOM), the feed material is in the form of sheets; the layer of specified contour is cut by applying laser on the specified path. The individual sheet acts as the layer and thus the thickness of the sheet becomes the layer thickness. As the printing proceeds the layers are thermally bonded together to fabricate the part. The cutting of the sheet at the specified contour is performed using a laser. The ultrasonic additive manufacturing is the only technology in available 3D printing systems that use metallic sheets and do the fabrication at low temperatures. The fabricated part is then taken to a CNC machine for postprocessing. The leftover material from the sheet can be recycled for future use. It has been observed that the surface quality produced from LOM is inferior when compared against the powder bed fusion technology. Nevertheless, the cost of raw material, as well as the printing cost, is relatively low (Blok et al., 2018; Chang, 2015; Gurr & Mu¨lhaupt, 2012). Fused deposition modeling (FDM) Thermoplastics such as ABS, PLA, and PC are the material employed in the process of FDM. In the fabrication of part using this technology, a solid filament of plastic is melted to semisolid paste and extruded through the nozzle to deposit on the specified contour layer by layer. This process is widely available and economically viable. A lot of beginners use this method to develop their understanding on rapid manufacturing technology using this system (Chitresh, Singh, & Himanshu, 2014). The influential parameters in this technology include the layer thickness, printing orientation, raster width, air gap, raster angle nozzle diameter, heating temperature, and printing speed. Researchers have developed parts by using multimaterials employing more than one printing nozzles having filaments of different materials (Wang et al., 2017). The details about which are discussed in the forthcoming sections. Fig. 13.8 represents the schematic diagram of the fabrication process using FDM. Ease of operation in FDM has made this technology viable for the application in both commercial and research projects, in the field of biomedical and tissue engineering. Stereolithography Stereolithography (SLA) is the oldest RP process among all the processes mentioned in this chapter. In this process, photocurable polymer resins are employed for the development of the part; most frequently used resins are acrylic or epoxybased monomers. These monomers solidify under the exposure of UV radiation. The monomers bond to become polymer at the instant they are exposed under radiation. After this the platform descends and liquid monomer covers up the polymer layer for the formation of the next layer. The researchers have incorporated particles of ceramics to produce a ceramic-filled composite by using this process. The influential parameter in this process is the energy of the incident UV



CHAPTER 13 Rapid prototyping

FIGURE 13.8 Schematic of the FDM: (1) vertically movable platform; (2) horizontally movable, heated deposition unit with nozzles; (3) model material; (4) support material; (5) feedstock of filament rolls (Gurr & Mu¨lhaupt, 2012).

FIGURE 13.9 (A) Part-building process, (B) the iPro 9000 XL system (, and (C) dashboard built by using iPro 9000 XL (Chang, 2015).

beam which can even alter the thickness of the developed layer. However, the part accuracy, in this process, is very good because of which this technology finds wide application in the field ofnanocomposites. It can be employed to print parts up to a resolution of 10 µm (Chang, 2015; Chua et al., 2020; Ngo et al., 2018; Wang et al., 2017). Fig. 13.9A shows the part building process of SLA. The other parts of the same figure, that is (B) and (C), show the actual image of iPro 9000 XL and a dashboard fabricated from the same printer, respectively.

13.4 Applications

FIGURE 13.10 A schematic view of stereolithography process: (1) photopolymerizable resin, (2) movable platform in the vertical direction, (3) CO2 laser, (4) scanning resin surface by optical systems, and (5) horizontal wiping blade (Gurr & Mu¨lhaupt, 2012).

A renowned company in this field is 3D systems that offer a variety of printers, with different configurations like SLA viper, iPro 8000MP, iPro 9000 XL, etc.; the price range typically varies between $180K and $950K. The one shown in the above picture is iPro 9000 XL which is the largest platform that is widely used for commercial applications across the world. The dimension up to which it fabricates the parts can be as big as 1500 mm 3 750mm 3 550 mm (59.1 in. 3 29.53 in. 3 21.65 in.). Products like an entire dashboard or complete bumpers can be fabricated by this printer. Fig. 13.10 shows a schematic representation for the working principle involved in the photopolymerization technique. In Fig. 13.11, a demonstration of the remaining RR processes is pictorially represented to enhance the imagination of the reader. Table 13.5 describes the contemporary RR systems detailing about physical form of feed materials, involved working principal, employed material, resolution, and the associated advantages and disadvantages. The surface roughness of the fabricated part is reported in Table 13.6; a quantitative comparison can thus be performed on the surface quality that can be obtained from different types of 3D printing systems. The associated layer thickness is also reported in the table for each of the system.

13.4 Applications RP technology finds a wide variety of applications in various sectors of engineering and biomedical. Development of complicated design for molds, dental applications,



CHAPTER 13 Rapid prototyping

FIGURE 13.11 Schematic representation of a typical (A) FDM setup, (B) 3DP setup, (C) SLA setup, (D) SLS setup, and (E) 3D plotting setup (Wang et al., 2017). SLA, Stereolithography; SLS, selective laser sintering; 3DP, three-dimensional printing.

fabrication of soft tissue in medical application, prosthetics, etc. has been made remarkably simple by the use of 3D printers. These parts required a complex and expensive process to develop and hence were far from the reach of people belonging to a relatively weaker economic section of the society. In addition to this, the effectiveness, accuracy, and surface finish achieved in fabrication of intricate and complex geometry form found to be superior in performance when compared to conventional methods. Apart from evident advantages like fabrication flexibility and customization the RP technology does not cause any wastage of the raw material. Also, the printers do not require any dedicated infrastructure or similar facility for operating (Tiwari, Pande, Agrawal, & Bobade, 2015; Chitresh, Singh, &

Table 13.5 Contemporary rapid prototyping techniques in summarized form (Wang et al., 2017).


State of starting of materials



Extrusion and disposition


Liquid or paste



Pressurized syringe extrusion, and heat or UVassisted curing Drop-on-demand binder printing




Liquid photopolymer

Working principle

Heat-induced sintering and laser scanning Laser scanning and UV-induced curing

Typical polymer materials Thermoplastic, like PC, ABS, PLA, and nylon PCL, PLA, hydrogel

Any material can be supplied as powder, binder needed Polymer powder and PCL Photocurable resin (epoxy acrylate-based resin)

Resolution (Z-direction, µm)



50 200 (Rapide Lite 500)

Low cost, multimaterial, good strength capabilities

5 200 (Fab@home)

High printing resolution, soft material capability

Nozzle clogging, anisotropy Low mechanical strength, slow

100 250 (Plan B Ytec3D)

Material capabilities, low cost, multi-material capability, easy removal of support powder Good strength, easy removal of support powder High-resolution printing

80 (Spo 230 HS)


Clogging of binder jet, binder contamination High cost, powder surface Material limitation, high cost, cytotoxicity

ABS, Acrylonitrile butadiene styrene; PC, polycarbonate; PLA, polylactic acid; PCL, polycaprolactone, SLA, stereolithography; SLS, selective laser sintering; UV, ultraviolet; 3DP, three-dimensional printing.


CHAPTER 13 Rapid prototyping

Table 13.6 Layer thickness and surface roughness (Kumbhar & Mulay, 2018). S. No.

Name of process

Surface roughness (Ra), (µm)

Minimum layer thickness (mm)

1 2

3D printing Poly-jetting process LOM SLA FDM SLS

12 27 3 30

0.175 0.10

6 2 9 5

0.114 0.10 0.254 0.125

3 4 5 6

27 40 40 35

LOM, Laminated object manufacturing; SLA, stereolithography; SLS, selective laser sintering.

Himanshu, 2014). Collectively considering all these advantages of the technology finds a wide application in various fields as mentioned below: •

Medical science and bioengineering applications

The scope of 3D printing is increasing in various sectors of the technology but the development and the benefits reaped in the field of medical are remarkable. The development in this field is further divided into following subcategories: 1. Rehearsal and planning before surgery There are many examples where 3D printed replicas of internal organs have assisted in the efficient planning of surgery. But the most eye-catching example is the surgery that separated two conjoined twins. The twins were born with joined heads; hence surgery was required to separate their heads. The conventional approach of planning that involved two-dimensional pictures from X-ray and computed tomography (CT) scans was producing a surgery that required nearly 97 h to complete. Working on a surgery that will last this long was very overwhelming; also the blood supply for these many hours was not possible. Using the data from X-rays and CT scans a 3D printed replica of the conjoined head was fabricated. The surgeons planned the surgery by studying the printed model. The surgery took only 22 hand was a success.1 Other examples involved the development of replicated models for surgery planning provided by different authors. Another example is the Sandia Lab replica of the human spine (Chang, 2015) (Fig. 13.12). 2. Educational purposes Educational institutes develop physical 3D printed models of internal body organs to make visualization simpler so that the student mind can appreciate the concept. 1

13.4 Applications

FIGURE 13.12 Replica of the human spine for planning a surgery at Sandia Labs (Chang, 2015).

3. Dental application Use of the RR technology in surgery planning for a dental implant is also very common. 3D printing is also employed for the fabrication of crowns for teeth. Chang (2015) quotes the significance of RP technology from the anthropological point of view. If there are only one or two specimens the entire teeth can be printed using 3D printing. 4. Prosthesis customization and implants Chitresh, Singh, & Himanshu, (2014) have employed 3D printing from the fused deposition modeling (FDM) for the customization and printing of prosthetic implants. This study carried out by the author is an example of how the CAD file is directly obtained after scanning the required geometry at different orientations. 5. Tissues and scaffolds There are examples where researchers have tried and successfully printed tissue scaffolds. Sometimes these scaffolds require metals such as titanium because of their biocompatible nature in the human body (Chua et al., 2020). However, there are 3D printing methods that employ metal as raw material. Ngo et al. (2018) have cited a report from Wohlers that the biomedical market represents 11% of the total market share in the field of RR technology. According to the report in the year 2016, the total market share of RR globally is around $6.1 billion. The flexibility and ease of customization in 3D printing are very useful in the development of implants and tissues. These parts are produced faster and more easily (Ngo et al., 2018). Patient-specific implants are easy to produce as scanned models of required implants are easily accessible to the domain of doctors and the 3D printer can print the implant in almost no time. Doctors have also used replicated model of scanned body parts to plan a surgery which reduces the time of surgery and the associated risk to the health of the patient (Fig. 13.13).



CHAPTER 13 Rapid prototyping

FIGURE 13.13 (A) 3D CAD, computer aided drafting model individuates a defect in the mandible from CT, computed tomography scan images; (B) motion distribution of print from the developed software. Green, blue, and red indicates the paths of PCL, polycaprolactone, Pluronic F-127, and hydrogel, respectively. (C) AM, additive manufacturing process; (D) 3D printed bony defect implant, cultured in osteogenic medium for 28 days; (E) osteogenic differentiation confirmed by Alizarin Red S staining, showing calcium deposition. (Ngo et al., 2018). .

• Aerospace 1. The aerospace industry shares approximately 18.2% of the total market share which is as per the Wohlers report cited by Ngo et al. (2018). 2. Building protective (Blok et al., 2018; Chang, 2015; Chua et al., 2020; Ngo et al., 2018; Wang et al., 2017).

13.5 Advancements in the rapid prototyping technology Since the development of RR techniques, there have been numerous advancements that can be classified into two broad categories:

13.5 Advancements in the rapid prototyping technology

13.5.1 Improvement of product quality Researchers working on this platform intend to address the shortcomings associated with 3D printing, which include distortion, part warping, dimensional accuracy, etc. To reduce these shortcomings, the influential parameters like nozzle size, printing speed, and melting temperature are addressed. Different researchers across the world have studied these parameters individually to investigate their degree of dependence on the product quality. Efforts are being made to optimize the working conditions of these parameters to improve the part quality (Blok et al., 2018; Chang, 2015; Chua et al., 2020; Gurr & Mu¨lhaupt, 2012).

13.5.2 Improvement on versatility of rapid prototyping Initially, limited materials were used in RP technology; with the advancement in the systems more materials are now used. Over the period of time, researchers have expanded the capabilities of 3D printing technology and the expansion is still going on. In the present scenario, biocompatible materials that can be used in prosthetics and dental applications are developed (Giachini et al., 2020; MacDonald & Wicker, 2016; Matsuzaki et al., 2016; Ngo et al., 2018). The development of the parts with intricate geometry from the traditional manufacturing systems is a complex job and the outcome is not as effective. Also, there was a considerable amount of material loss that increased the cost of production. That loss has been minimized specially for complex shapes which are now easily fabricated by additive manufacturing, also referred to as rapid prototyping. This has now inspired the investigators to print a fully functional system assembled directly from a 3D printer. The upcoming generation of 3D printing is expected to have the capability to directly fabricate a fully functional working component in a single run of the process. There are works done by researchers in different parts of the world that have been covered by a literature study conducted by MacDonald and Wicker (2016). There is a paradigm shift in this approach where the objective of the process will be to fabricate in contrast to the previous system of assembly. This process will be a nonassembly system producing a multifunctional end-use device. According to the chief executive officer of General Electrical, Jeff Immelt, there is an ambitious goal of producing as many as 100,000 jet engines to meet the target demand. To achieve this level of production the organization has planned to invest a sum of 3.5 billion dollars in the technology of additive manufacturing. Most of the 3D printing patents are also expiring, resulting in the expansion of this technology by the entry of new manufacturing companies employing the technology of 3D printing (MacDonald & Wicker, 2016). There are several other examples in which the research and development departments of various companies have successfully achieved this target. Those examples are explained in the forthcoming section.



CHAPTER 13 Rapid prototyping

13.5.3 Multifunctional fabrication process When it comes to defining multifunctional fabrication process in rapid manufacturing technology, it can be described as an additive manufacturing process in which apart from the fabrication of any intricate shape an additional functionality is also introduced in the printed part. Along with this multicolor, density variation, stiffness gradient, etc. are embedded in the printed structure.

13.5.4 Printable and embeddable functions In addition to the basic shape printing, the next-generation 3D printing technology has evolved and became capable to embed certain types of functionality like transducing, functionally gradient, thermal management, energy storage, propulsion utility, and sensing, most of which are described as follows. Sensors In the production of sensors using RR technology, the researcher has put a considerable amount of approaches by focusing on the following two ways to achieve this target: firstly, interrupting the process and then embedding the sensor directly into the printed part; secondly, to arrange the setup such that the entire sensor can be directly printed along with the structure. A highly stretchable sensor employing the 3D printing known carbon-stretchable sensor is printing from a carbon-based ink with an elastomer structure (Muth et al., 2014). Shemelya et al. have demonstrated the development of a capacitive touch sensing system by employing a wire-on-wire submerged with the 3D-printed thermoplastic structure. This sensor can be used as a single capacitive plate for a touch sensor (Shemelya et al., 2014). Actuations Researchers across the world have printed several working models and prototypes of actuators. Prosthetic hands with embedded external motion have been printed. Richter and Lipson successfully printed a bio-inspired flapping wings insect by employing VAT polymerization (Richter & Lipson, 2011). Thermal management With further advancement in multifunctional 3D printing, advance thermal management systems are also developed. Complex large surface area structures with good thermal conductivity have been fabricated using metal 3D printing techniques (Wong, Tsopanos, Sutcliffe, & Owen, 2007). Researchers are hoping to produce an advanced system with embedded heat pipes and reservoirs for material with phase change. These fabricated systems lead to the improvement in the thermal management system of 3D structures (MacDonald & Wicker, 2016).

13.5 Advancements in the rapid prototyping technology Energy storage A study has been reported of printing a battery, setting up an example for the development of an energy storage system. Malone, Berry, and Lipson (2008) have successfully printed customizable arbitrary shaped battery system. This contained 3D printed structures after sintering (Malone et al., 2008). Antennas and electromagnetic structures Additive manufacturing offers few distinctive features that have increased its utility and application throughout various fields of technology. One of these distinctive features is the presence of provision to intentionally embed porosity within the fabricated part. This distribution of porosity can be varied throughout the structure resulting in variation of density within the structure. This feature functionally grades the permittivity and permeability of the structure. Developers have exploited this feature in enabling the electromagnetic transitions through the interface of the material and minimize reflections which in return can be used for sculpturing EM waves in antennas. Still, the unintentional porosity is difficult to avoid which acts as a challenge in 3D printing processes that involve thermoplastics (Deffenbaugh, Rumpf, & Church, 2013; Liang et al., 2014; Rumpf, Pazos, Digaum, & Kuebler, 2015). Propulsion Polymers manufactured from additive manufacturing deliver appropriate dielectric strength. While fine quality available copper wires possess low resistance sufficient enough to serve the purpose. Researchers were able to show this kind of propulsion by supplying high voltage in fabricated test coupons for igniting micro-pulsed plasma thrusters. This shows the utility of multipurpose additive manufacturing with wire and components placement, which benefits the manufacturing of space vehicles and their embedded component (Marshall et al., 2015).

13.5.5 Fiber-reinforced polymer composites There are several examples of the development of composite material using RR technology. These included various types of reinforcement material within the structure itself. Also, there are examples of FRC material printed from RP technology. But in most cases, the orientation of the fiber was not user-defined. However, there are examples where researchers have embedded the information about fiber orientation. Matsuzaki et al. have fabricated jute fiber-reinforced polymer composite using 3D printing technology. In the composite fabricated by them, the unidirectional alignment of jute fiber was made possible. The strength of the fabricated material was compared with one which was produced from the conventional fabrication process. The result of the comparison had shown superior performance of the unidirectionally aligned composite specimen fabricated



CHAPTER 13 Rapid prototyping

FIGURE 13.14 (A) Carbon fiber-reinforced polymer. (B) Jute fiber-reinforced polymer tensile test specimens. (C) Cross-sectional view and (D) magnified cross-section of the carbon fiber composite specimen (Matsuzaki et al., 2016).

from RR. Unidirectionally aligned carbon fiber-reinforced polymer composite was also fabricated and tested against their conventionally fabricated counterparts. Fig. 13.14 shows the pictures of the fabricated specimens (Matsuzaki et al., 2016). In the research work conducted by Le Duigou et al., PLA-based Flax FRC was 3D printed to investigate the tensile strength in longitudinal and transverse directions. The specimen showed superior magnitude of the properties, when compared against the composite prepared employing conventional methodology for fabrication (Le Duigou, Barbe´, Guillou, & Castro, 2019).

13.5.6 Functionally graded materials using rapid prototyping Giachini et al. used additive manufacturing of cellulose-based material with continuous, multidirectional stiffness gradient. This process is also termed multimaterial additive manufacturing. FDM process was employed for the development of this FGM containing two different materials. Various combinations of the materials were tested with base material such as hydroxyethylcellulose and different compositions of other materials like lignin, microfibrillated cellulose, citric acid, and hydrogen chloride. The composition gradient was embedded within the system producing a continuous stiffness gradient (Giachini et al., 2020).

13.5.7 Comparison with traditional manufacturing These systems are recently developed, and the user feedback of the product is not available, making it difficult to comment about reliability or durability.


However, when they are subjected to the specified test as per the standards they show comparable performance with that of traditional manufacturing techniques.

13.6 Conclusion Flexibility, freedom of design, and ease of prototyping have been redefined after the development of rapid manufacturing technology. This system has evolved significantly after it was experimented for the first time. Contemporary development in the field of RR suggests that the potential of this technology is remarkably high and might partially or fully substitute the conventional manufacturing technologies. The scope of research and development in this field is wide and it is still expanding. Commercialization of this technology has now started to gain pace and is rising exponentially. With the involvement of bioengineering application, development of multifunctional devices from 3D printing, fabrication of composite material, and FGMs this technology is not only on the path of representing the pioneer of interdisciplinary achievement in the field of engineering but will also produce lots of jobs and opportunity in the future benefiting the economy. The market share of the rapid manufacturing technology is also increasing at a high pace and it is expected that it will keep on increasing. With the commercialization of the technology soon the reliability of the products and parts fabricated will become clear by the feedback provided after these are used by the end customers. These reviews from the general customer will increase the scope of research, as well as applications. Along with the same, there is a high probability that soon this technology will become less expensive and will become available for the use of the public. With further advancement in 3d printing, we can get personal 3D printer in an affordable price in the market. While, STL file for useful good and stationary will be available online by the manufacturer making additive manufacturing as user friendly as conventional printing. As far as biomedical is concerned the accuracy and the capability of RP system to produce intricate geometry when clubbed the advancement in the development of biocompatible material, it is not unrealistic to expect a groundbreaking breakthrough in the field of medical science.

References Blok, L. G., Longana, M. L., Yu, H., & Woods, B. K. S. (2018). An investigation into 3D printing of fibre reinforced thermoplastic composites. Additive Manufacturing, 22, 176 186. Chang, K.-H. (2015). Chapter 14 Rapid prototyping. In K.-H. Chang (Ed.), e-Design (pp. 743 786). Academic Press, ISBN 9780123820389.



CHAPTER 13 Rapid prototyping

Chen, R. K., Jin, Y.-A., Wensman, J., & Shih, A. (2016). Additive manufacturing of custom orthoses and prostheses—A review. Additive Manufacturing, 12, 77 89. Chitresh, N., Singh, A., & Himanshu, C. (2014). Customised prosthetic socket fabrication using 3D scanning and printing. In Conference additive manufacturing society of India. Additive Manufacturing Society of India, Banglore. Chua, C. K., Leong, K. F., & An, J. (2020). Introduction to rapid prototyping of biomaterials. Rapid prototyping of biomaterials (pp. 1 15). Woodhead Publishing. Deffenbaugh, P. I., Rumpf, R. C., & Church, K. H. (2013). Broadband microwave frequency characterization of 3-d printed materials. IEEE Transactions on Components, Packaging and Manufacturing Technology, 3(12), 2147 2155. Available from https:// Giachini, P. A. G. S., Gupta, S. S., Wang, W., Wood, D., Yunusa, M., Baharlou, E., . . . Menges, A. (2020). Additive manufacturing of cellulose-based materials with continuous, multidirectional stiffness gradients. Science Advances, 6(8), eaay0929. Gurr, M., & Mu¨lhaupt, R. (2012). Rapid prototyping. Polymer science: A comprehensive reference (pp. 77 99). Amsterdam: Elsevier. Krishna, V., Kate, K. H., Satyavolu, J., & Singh, P. (2019). Additive manufacturing of natural fiber reinforced polymer composites: Processing and prospects. Composites Part B: Engineering, 174, 106956. Available from compositesb.2019.106956. Kumbhar, N. N., & Mulay, A. V. (2018). Post processing methods used to improve surface finish of products which are manufactured by additive manufacturing technologies: A review. Journal of The Institution of Engineers (India): Series C, 99(4), 481 487. Available from Le Duigou, A., Barbe´, A., Guillou, E., & Castro, M. (2019). 3D printing of continuous flax fibre reinforced biocomposites for structural applications. Materials & Design, 180, 107884. Liang, M., Ng, W. R., Chang, K., Gbele, K., Gehm, M. E., & Xin, H. (2014). A 3-D Luneburg lens antenna fabricated by polymer jetting rapid prototyping. IEEE Transactions on Antennas and Propagation, 62(4), 1799 1807. Available from https:// MacDonald, E., & Wicker, R. (2016). Multiprocess 3D printing for increasing component functionality. Science (New York, N.Y.), 353(6307). Malone, E., Berry, M., & Lipson, H. (2008). Freeform fabrication and characterization of Zn-air batteries. Rapid Prototyping Journal, 14(3), 128 140. Available from https:// Marshall, W. M., Stegeman, J. D., Zemba, M., MacDonald, E., Shemelya, C., Wicker, R., . . . Kief, C. (2015). Using additive manufacturing to print a CubeSat propulsion system. In 51st AIAA/SAE/ASEE joint propulsion conference (p. 4184). Matsuzaki, R., Ueda, M., Namiki, M., Jeong, T.-K., Asahara, H., Horiguchi, K., . . . Hirano, Y. (2016). Three-dimensional printing of continuous-fiber composites by innozzle impregnation. Scientific Reports, 6(1), 1 7. Muth, J. T., Vogt, D. M., Truby, R. L., Mengu¨c¸, Y., Kolesky, D. B., Wood, R. J., & Lewis, J. A. (2014). Embedded 3D printing of strain sensors within highly stretchable elastomers. Advanced Materials, 26(36), 6307 6312. Ngo, T. D., Kashani, A., Imbalzano, G., Nguyen, K. T. Q., & Hui, D. (2018). Additive manufacturing (3D printing): A review of materials, methods, applications and challenges. Composites Part B: Engineering, 143, 172 196.


Richter, C., & Lipson, H. (2011). Untethered hovering flapping flight of a 3D-printed mechanical insect. Artificial Life, 17(2), 73 86. Rumpf, R. C., Pazos, J. J., Digaum, J. L., & Kuebler, S. M. (2015). Spatially variant periodic structures in electromagnetics. Philosophical Transactions of the Royal Society A—Mathematical, Physical and Engineering Sciences, 373(2049). Available from Shemelya, C., Cedillos, F., Aguilera, E., Espalin, D., Muse, D., Wicker, R., & MacDonald, E. (2014). Encapsulated copper wire and copper mesh capacitive sensing for 3-D printing applications. IEEE Sensors Journal, 15(2), 1280 1286. Tiwari, S. K., Pande, S., Agrawal, S., & Bobade, S. M. (2015). Selection of selective laser sintering materials for different applications. Rapid Prototyping Journal, 21, 630 648. Wang, X., Jiang, M., Zhou, Z., Gou, J., & Hui, D. (2017). 3D printing of polymer matrix composites: A review and prospective. Composites Part B: Engineering, 110, 442 458. Wong, M., Tsopanos, S., Sutcliffe, C. J., & Owen, I. (2007). Selective laser melting of heat transfer devices. Rapid Prototyping Journal, 13(5), 291 297. Available from https:// Zindani, D., & Kumar, K. (2019). An insight into additive manufacturing of fiber reinforced polymer composite. International Journal of Lightweight Materials and Manufacture, 2(4), 267 278. Available from ijlmm.2019.08.004.



Self-assembled polymer nanocomposites in biomedical applications


Anurag Dutta1,2, Manash Jyoti Baruah1, Satyabrat Gogoi2 and Jayanta Kumar Sarmah2 1

Department of Chemical Sciences, Tezpur University, Tezpur, Assam, India Department of Chemistry, School of Basic Sciences, The Assam Kaziranga University, Jorhat, Assam, India


14.1 Introduction Nanocomposites materials are those materials where one or more of the primary constituent phases has a dimension in the nanoscale range. Owing to widespread applicability and the tunability, they have been the center stage of attraction in the academia as well as industrial research. The reason behind this is the ability of such materials to inherit the advantageous properties of the constituent components and/or enhance it toward being a multifunctional entity. The nanocomposites in polymeric forms have offered a plethora of applications with their inherence quality of target specific designing and the ability to meet the demands of functional material world. Polymer nanocomposites usually contain an inorganic nanomaterial (usually particles, tubes, and/or wires of nanometric dimensions, or nanoclay) decorated within/over an organic matrix (polymers or biomacromolecules). The synergy between the characteristics of the inorganic component and the organic polymer aids such nanocomposite materials in showing amplified optical, mechanical, magnetic, thermal, and optoelectronic properties (Kumar & Jouault, 2013). Such synchronism and display of diversified properties have resulted in such polymer nanocomposites, finding indispensable utilities in the field of sensing (Hosu, Barsan, Cristea, S˘andulescu, & Brett, 2017), designing of solar cells (Zhao & Lin, 2012), catalysis (Marcoux, Florek, & Kleitz, 2015), electronics (Yousefi et al., 2014), biotechnology (Wang, Cui, Wang, & Li, 2016; Wang, Yang, et al., 2016), and biomedicine. The surge in the innovations in polymer chemistry and nanofabrication technologies has not only pushed the boundaries of research in the field of designing newer types of polymer nanocomposites but also in the production of multifunctional materials. This has opened an arena for the development and production of polymer nanocomposites with tailored functionalities for applications which are highly sophisticated and efficient in nature. However, this Advances in Biomedical Polymers and Composites. DOI: © 2023 Elsevier Inc. All rights reserved.



CHAPTER 14 Self-assembled polymer nanocomposites

achievement is highly dependent upon the synthetic methodologies employed and much has already been achieved (Li et al., 2016; Matsuura, 2017; Nie, Li, Wang, & Zhang, 2016). Among the available protocols, self-assembly is one such technique that has been extensively used. The reason being the simplicity and economic efficacy of the protocols as well as the precision and the flexibility associated with them. Self-assembly is a process of spontaneous molecular arrangement of disordered entities of molecules into well-defined ordered structures via local interactions among the constituent entities themselves. Major approaches that have been reported, but not limited to, for the construction of self-assembled polymer nanocomposites are: 1. Surface modification of grafted polymer (Kumar, Bansal, Behera, Jain, & Ray, 2016; Kumar, Behera, Thakre, & Ray, 2016) 2. Spin coating (Nunes-Pereira et al., 2015) 3. Deposition (Hu et al., 2012) 4. Layer-by-layer assembly (Zhang, Tong, & Xia, 2014) Given to the widespread liberty that can be taken toward the designing of selfassembled polymer nanocomposites, the utility and employment into biomedical applications are expanding at a very healthy pace (Ahmad, Manzoor, Singh, & Ikram, 2017; Komiyama, Yoshimoto, Sisido, & Ariga, 2017; Yi, Zhang, Webb, & Nie, 2017). The foundation to this endeavor is based upon the never ending list of self-assembled biological systems that form the very basis of life alone. Be it the construction of cellular membranes via assembling the phospholipid bilayers, protein folding, or the characteristic double-helix structure of the DNA, they are all types of biological self-assembly. In fact, they can also be called as biological self-assembled nanostructures. The ligand-to-receptor interactions that form the basis of neural signal transfer and enzyme catalysis in the biological systems can also be attributed to self-assembly of the complex biological polymer nanostructures. Self-assembly also accounts for the formation of molecular crystals, various forms of colloids, miscelles, self-assembled monolayers, and phase-separated polymers. The point to note here is that such type of molecular self-assembly is the key to the emergence of life and its maintenance. Concepts for modern applications of self-assembled polymer nanocomposites have been derived from synthetic amino acids, oligo- and polypeptides, dendrimeric ensembles, polymers and pi-conjugated compounds toward construction of various nanostructures like nanotubes, fibers, micellar aggregations, and vesicles. In addition to this, people have also considered small-molecule self-assembly as building units of structurecontrolled materials. In a similar way, DNA-based nanomaterials have written their own story of being potential diagnostics and drug delivery tools. In the beginning of the 21st century, (Whitesides & Boncheva, 2002) mentioned that newer nanomaterials will find a new face with the process of self-assembly. This was based on grounds that the process is not only important toward maintaining the standard of living or for technological advancement of mankind, but also for

14.2 Methods of preparation

FIGURE 14.1 Schematic representation of the subject of this chapter: preparation of self-assembled polymer nanocomposite and their biomedical applications.

keeping it alive. Living materials such as the cell contain complex nanostructures such as the lipid membranes, protein aggregates, complex molecular machines such as the folded proteins and the nucleic acids, etc. These have shown the natural tendencies of self-assembly. Now, it can be said that self-assembly has paved a way into a diversified arenas and has provided ample opportunities toward the development of novel functional materials and building blocks of life through a close-knit exchange of concepts among them. In this chapter we shall see in detail the ways in which self-assembled polymer nanocomposites are formed and their various applications in biomedical sciences (Fig. 14.1).

14.2 Methods of preparation of self-assembled polymer nanocomposites The self-assembly process involves a fine-tuned balance between the attractive and repulsive forces, which commences the aggregation, along with an entity that gives a proper direction to the growth of the polymer nanocomposite (Fig. 14.2).



CHAPTER 14 Self-assembled polymer nanocomposites

FIGURE 14.2 The distinct forces involved in self-assembly.

While self-assembled nanocomposites can be constructed by using a number of available methods, designing self-assembled polymer nanocomposites require extra attention and complexity is involved in the due course of its formation. Two methods that have risen above others in terms of efficacy, sustainability, and ease of follow-up are polymer grafting on or from modified surface of nanoparticles and layer-by-layer assembly. The methods are briefly described below:

14.2.1 Polymer grafting on/from the modified surface of nanoparticles The technique of polymer grafting for modifying the surface of nanoparticles has been known for quite a while now (Zoppe et al., 2017). This has allowed the uniform dispersion of the nanoparticles in to the matrix of the polymer at a nanometer scale. Polymers with well-defined structural units and molecular weight along with a small polydispersity ratio are synthesized via controlled radical polymerization. Among the radical polymerization methods, atom transfer radical polymerization, well known as ATRP, is a technique that offers an efficient route toward preparing multifunctional composites with end group functionality (Siegwart, Oh, & Matyjaszewski, 2012). Another specialized form of ATRP is surface-initiated ATRP (SIATRP) (Fig. 14.3). This technique allows the polymer growth from the solid surface and is known to show several advantages in the preparation of the mentioned class of materials. The advantages associated with this type of polymerization are: 1. Ability to produce polymer chains which are covalently grafted on the solid surface. Such polymer chains can then control the functional properties of the entire nanocomposite like the interfacial properties, concentration of the polymer, and also regulate the yield of the nanocomposite synthesized.

14.2 Methods of preparation

FIGURE 14.3 Schematic representation of surface-initiated polymerization.

2. The molecular weight of the nanocomposite, its polydispersity ratio, and the composition can be tailored with considerably good control. 3. The applicability of the method to a variety of monomers under varying conditions is one of the foremost advantages of the protocol. This provides a platform to designing the substrate particles with varying functionalities. Until now, the SIATRP technique has been successfully studied with nanomaterials such as silica (Mao et al., 2017), multiwalled carbon nanotubes (Song et al., 2016), graphene and graphene oxide (GO; Ata, Banerjee, & Singha, 2016), gold (Lee, Kim, Park, Cho, & Choi, 2016), magnesium hydroxide (Liu, Feng, Chang, & Kang, 2012), and clay (Vo et al., 2016). A fine example of this technique was demonstrated by Huang et al. (2017), where ultraviolet light, in the presence of an organic catalyst, 10-phenylphenothiazine, was used to induce polymerization for modifying the surface of mesoporous silica nanoparticles (MSNs) with itaconic acid (IA) and polyethylene glycol methylacrylate (PEGMA) (Fig. 14.4). This was a novel metal-free ATRP, and the polymer nanocomposite [MSNs-NH2-poly (IA-co_PEGMA)] thus formed demonstrated a very good dispersity in both aqueous and organic media. Moreover, it was used as a potential carrier for the drug cis-platin.

14.2.2 Layer-by layer assembly technique The method based upon sequential deposition of oppositely charged species is broadly termed as the layer-by-layer assembly technique. Originally developed for polyelectrolyte systems, this technique has now found applications in almost all types of polymer growth protocols and with almost any type of component. Due to its broad scope of applicability, simplicity, versatility, and robustness, it has been accepted as a go-to choice for the synthesis of polymer nanocomposites (Ariga et al., 2014; Cui, Li, & Decher, 2016; Cui, Yang, Wang, & Wang, 2016; Xuan et al., 2017). There has been a variety of nanocomposites with diverse



CHAPTER 14 Self-assembled polymer nanocomposites

FIGURE 14.4 Schematic route for the synthesis of MSNs-NH2-poly(IA-co-PEGMA) via a metal-free ATRP. Reproduced with permission from Huang, L., Liu, M., Mao, L., Xu, D., Wan, Q., Zeng G., et al. (2017). Preparation and controlled drug delivery applications of mesoporous silica polymer nanocomposites through the visible light induced surface-initiated ATRP. Applied Surface Science, 412571 412577. 10.1016/j.apsusc.2017.04.026.

applications which were developed with the help of this technique. Some of them are summarized in Table 14.1. This technique is capable of fabricating diversified multifunctional polymer nanocomposites with ultra-high precise dimensional properties. Moreover, the content and dispersion can also be fine-tuned to a large extent. Ma, Cai, Qi, Kong, and Wang (2013) constructed a nanocomposite, which comprised of polyacrylic acid (PAA) functionalized graphene bearing poly(diallyldimethylammonium chloride) protected Prussian blue (PDDA-PB) nanoparticles (Fig. 14.5). This nanocomposite was used as a hydrogen peroxide sensor. The graphene and PB nanoparticles expressed a synergistic effect and catalytically reduced H2O2. The response toward the change in concentration of the peroxide was quick and the steady-state signal was reached within 2 s. The excellence in the properties demonstrated could be attributed to the increase in the rate of electron transfer between the electrodes and the detection molecules due to the large surface to volume ratio of the nanomaterial that was deposited on the electrode. This finally led to rapid and highly sensitive current response. This work is a noteworthy example of bio-sensing. The salient feature of this technique is that the electrocatalytic activity of the film could be designed and tuned by simply selecting the number of bilayers required or by choosing the electrically active species. Another case of layer-bylayer assembled GO nanocomposite films used in increasing the mechanical properties of poly(allylamine hydrochloride) and poly(sodium 4-styrene sulfonate) containing polyelectrolyte multilayer films, where the fibroblast cell adhesion and

14.2 Methods of preparation

Table 14.1 Some polymer nanocomposites synthesized via layer-by-layer assembly technique. S. No.

Type of polymer nanocomposite


Tricobalt tetroxide (Co3O4)/poly(styrene sulfonate) Graphene oxide/poly (allylamine hydrochloride) Polypyrrole/titanium dioxide (TiO2) Halloysite/polyaniline Nanoclay/ polyethylenimine


3 4 5



Humidity sensor

Zhang, Jiang, Sun, and Zhou (2017)

Biointerface with excellent mechanical properties Gas sensor

Qi, Xue, Yuan, and Wang (2014), Qi, Yuan, Yan, and Wang (2014) Cui, Yang, et al. (2016), Cui, Li, et al. (2016) Huang et al. (2016) Ziminska, Dunne, and Hamilton (2016)

Supercapacitor Foam coating agent with customizable properties

FIGURE 14.5 Scheme for assembling process of (PAA graphene/PDDA PB)n multilayer films. Reproduced with permission from Ma, J., Cai, P., Qi, W, Kong, D, & Wang, H. (2013). The layer-by-layer assembly of polyelectrolyte functionalized graphene sheets: A potential tool for biosensing. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 426, 6 11. colsurfa.2013.02.039.



CHAPTER 14 Self-assembled polymer nanocomposites

cell proliferation were studied, happens to be a beautiful example of biomedical and biotechnological advancement of assembled polymer nanocomposites. It will be quite a valid point to state that this highly tuned technique of engineering polymer matrix via incorporation of nanomaterials is an effective way of altering the physicochemistry of polymer composites and it holds promising contributions toward designing biologically active materials. In the following sections we shall discuss the applications of self-assembled polymer nanocomposites in biomedical sciences:

14.3 Applications