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Enables advanced tissue regeneration approaches via expertise from the fields of materials science and biology
Stimuli-Responsive Materials for Tissue Engineering comprehensively reviews the use of stimuli-responsive materials in the context of advanced tissue engineering approaches, highlighting applications, challenges, and solutions and reporting on the current state of the art of smart and multifunctional materials being used for tissue engineering, focusing on material types and their properties.
The progress that has already been achieved in the field is put into perspective by covering the remaining challenges in the research field of tissue engineering, and solutions are outlined to overcome those. By addressing challenges and ways to overcome them, Stimuli-Responsive Materials for Tissue Engineering is a highly practical resource on advanced tissue regeneration.
Stimuli-Responsive Materials for Tissue Engineering contains information on:
- Smart and multifunctional materials for tissue engineering, covering electroactive and magnetoactive materials
- Shape memory, photo-responsive, and controlled degradation of stimuli-responsive materials
- Tissue regeneration strategies based on smart and active biomaterials, covering bone, heart, and neural tissue regeneration
- Main applications where these biomaterials can be applied, such as in bone, muscle, and skin regeneration
- Other potential areas where the covered biomaterials are expected to make a major impact in the next decade
With comprehensive coverage of the subject, Stimuli-Responsive Materials for Tissue Engineering is an essential resource for materials scientists, bioengineers, engineering scientists, and biotechnologists seeking to understand advanced tissue regeneration approaches, current challenges, and potential solutions to advance progress in the field.
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Veröffentlichungsjahr: 2024
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
Preface
Part I: Smart and Multifunctional Materials for Tissue Engineering
1 Electroactive Materials for Tissue Engineering
1.1 Introduction
1.2 Relevance of the Electrical Signals in the Human Body
1.3 Relevance of the Electrical Signals in Cell Processes
1.4 Types of Electroactive Materials
1.5 Relevance of the Material’s Architecture
1.6 Final Remarks
Acknowledgments
References
2 Piezoelectric, Triboelectric and Magnetoactive materials for Tissue Engineering
2.1 Introduction
2.2 Piezoelectric, Triboelectric, and Magnetoactive Materials in Tissue Engineering
2.3 Summary and Conclusion
References
3 Shape-memory Polymers for Tissue Engineering Applications
3.1 Introduction
3.2 Shape-memory Effect in Polymers
3.3 Shape-memory Mechanism and Molecular Structure
3.4 SMP Triggered by Stimuli Different to Temperature
3.5 Requirements of SMPs as Scaffolds
3.6 Tissues-Specific Requirements
3.7 Shape-memory Homopolymers and Copolymers for Tissue Engineering
3.8 Shape-memory Composites for Tissue Engineering
3.9 Shape-memory Hydrogels for Tissue Engineering Applications
3.10 Conclusions and Future Trends
References
4 Photoresponsive Materials for Tissue Engineering
4.1 Introduction
4.2 Photoisomerizable Materials
4.3 Photopolymerizable Materials
4.4 Photocleavable Materials
4.5 Photothermal Materials
4.6 Conclusions and Outlook
Acknowledgments
References
5 Controlled Degradation of Stimuli-Responsive Materials for Tissue Engineering
5.1 Introduction
5.2 Degradation Mechanisms
5.3 Degradation Measurement Techniques
5.4 Conclusions and Future Perceptions
References
Part II: Tissue Regeneration Strategies Based on Smart and Active Biomaterials
6 Active Biomaterials for Bone Tissue Regeneration
6.1 Introduction
6.2 Ultrasound-Based Strategies for the Engineering of Biomaterials for Bone Regeneration
6.3 Magnetic Forces to Assist Biomaterials in Bone Formation
6.4 Piezoelectric Materials as Biomimetic Substrate for Skeletal Engineering
References
7 Muscle and Heart Tissue Regeneration Based on Stimuli-Responsive Materials
7.1 Introduction
7.2 Skeletal and Cardiac Muscle Organization
7.3 Stimuli-Responsive Materials for Muscle Tissue Engineering
7.4 Conclusions and Future Perspectives
Acknowledgments
References
8 Neural Tissue Regeneration Based on Stimuli-Responsive Materials
8.1 Introduction
8.2 Electroactive Smart Polymers: An Overview
8.3 Electroactive Smart Polymers for
In Vitro
Neuronal Outgrowth and Differentiation
8.4 Electroactive Smart Polymers as a Regenerative Approach for Neural Regeneration
In Vivo
8.5 Major Challenges and Future Directions
8.6 Final Remarks
References
9 Skin Tissue Regeneration Based on Stimuli-Responsive Materials
9.1 Introduction
9.2 Structural Organization of the Skin
9.3 Physiology of Wound Healing
9.4 Biomaterials for Skin Regeneration
9.5 Stimuli-responsive Materials
9.6 Future Perspectives
Acknowledgments
References
10 Cancer Therapies Based on Stimuli-Responsive Materials
10.1 A Brief Review
10.2 Chemotherapy and the Role Played by Stimuli-Responsive Materials
10.3 Stimuli Types and Targeting
10.4 Concluding Remarks and Perspectives
Acknowledgments
References
Summary and Outlook
Acknowledgments
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 Overview of
in vitro
and
in vivo
electrical stimulation effects on...
Table 1.2 Overview of relevant conductive materials for TE applications.
Table 1.3 Overview of relevant piezoelectric materials for TE applications....
Chapter 2
Table 2.1 Magnitude of piezoelectric coefficients corresponding to the vario...
Table 2.2 Potential range for various tissues in human body.
Table 2.3 Biocompatibility of selected piezoelectric materials for tissue en...
Table 2.4 Biocompatibility of selected triboelectric materials for tissue en...
Table 2.5 Biocompatibility of selected magnetoactive materials for tissue en...
Chapter 3
Table 3.1 Summary of some reported works about shape-memory homopolymer/copo...
Table 3.2 Example of shape-memory polymer composite (SMPC) scaffolds for tis...
List of Illustrations
Chapter 1
Figure 1.1 Electric activity in the human body.
Figure 1.2 Correlation between changes in Vm and intracellular calcium conce...
Figure 1.3 Possible intracellular responses to electrical stimulation. Membr...
Figure 1.4 (a) Band gap theory of conductors, semiconductors, and insulators...
Figure 1.5 Piezoelectric effect in quartz.
Figure 1.6 Magnetoelectric effect using (a) magnetostrictive particles or (b...
Figure 1.7 Thermoelectric materials: (a) Seebeck and (b) Peltier effects.
Figure 1.8 Representative SEM images of the distinct structures/morphologies...
Chapter 2
Figure 2.1 Introduction to scaffolds properties, preparations, and applicati...
Figure 2.2 Basic working mechanism of a piezoelectric device. Demonstration ...
Figure 2.3 Working modes of a TENG. (a) contact separation mode. (b) linear ...
Figure 2.4 Triboelectric series and demonstration of the combination of mate...
Figure 2.5 Basic working mechanism of a magnetoactive device. (a) Multiple l...
Figure 2.6 Important properties of the scaffolds require for tissue engineer...
Figure 2.7 Working mechanism of electrospinning. (a) Demonstration of real-l...
Figure 2.8 Working mechanism of electrospinning. (a) Demonstration of charge...
Figure 2.9 (a) Schematic representation of the chain...
Figure 2.10 Overall cell morphology of hMSCs analyzed by SEM after four days...
Figure 2.11 SEM images of the cultured Saos-2 cells on: (a,b) hydrophobic an...
Figure 2.12 (a) Percentage of activated cells grown on different PVDF scaffo...
Figure 2.13 (a) TEM image of the PVDF-TrFE/BNNT film showing curvilinear ele...
Figure 2.14 (a,b) Bright field microscopy images of Alizarin Red stained SaO...
Figure 2.15 (a) SEM images of electrospun PLLA.(b) SEM micrographs of hF...
Figure 2.16 (a) Scaffold-guided approach for TE. Cells are expanded ex vivo ...
Figure 2.17 (a) Macroscopic image of PVDF-TrFE scaffold used for cell seedin...
Figure 2.18 (a) FE-SEM micrograph of synthesized PANI nanoparticles, which r...
Figure 2.19 (a,b) SEM images of the linear microstructures on Al film and na...
Figure 2.20 (a) Schematic representation for the synthesis of CPDA hydrogel....
Figure 2.21 (a) Enlarged illustration of the device components, key material...
Figure 2.22 (a) Schematic illustration showing the structural design of the ...
Figure 2.23 SEM images of (a) PHB, (b) PHB/gelatin, (c) PHB/gelatin/Fe
3
O
4
....
Figure 2.24 (a) Cytotoxicity of pure PHB, PHB/gelatin, PHB/gelatin/Fe3O4 sca...
Figure 2.25 (a) FESEM images of PVDF-CFO microspheres.(b) Cross-section ...
Chapter 3
Figure 3.1 Scheme of the polymer chemical structures that driven by stimuli ...
Figure 3.2 Schematic representation of the shape-memory cycle composed of a ...
Figure 3.3 Schematic representation of the molecular structure requirements ...
Figure 3.4 General mechanism of SMPs based on reversible interactions like s...
Figure 3.5 Schematic representation of the external stimuli that can endorse...
Figure 3.6 Schematic representation of the followed strategy to prepare enzy...
Figure 3.7 Scheme illustrating the general steps of scaffold-based strategie...
Figure 3.8 Requirements for SMPs development for tissue engineering.
Figure 3.11 Generally employed inorganic filler in the preparation of SM com...
Figure 3.9 (a) Molecular structure of PLA, PEG, and photo-crosslinkable cinn...
Figure 3.10 (a) Implantation of PCL and aniline trimer PU scaffolds on rat c...
Figure 3.12 (a) Development of porous SMPC scaffolds (PCL/HA) by mixing HA p...
Figure 3.13 Effect HA concentration in PCL/HA scaffolds. PCLHA0 presents pur...
Figure 3.14 Monitoring of osteogenic differentiation of C2C12 myoblasts on d...
Figure 3.15 Effect of Cu
2+
and SiO
4
4−
released by CaCuSi4O10 (CUP)...
Figure 3.16 Macroscopic observation of DCol and Col scaffolds implanted in r...
Figure 3.17 Establishment of a cartilage defect model and macroscopic examin...
Figure 3.18 After 7 days of culture, immunostaining of cell hydrogel in diff...
Chapter 4
Figure 4.1 (a) Schematic representation of the photoisomerization process of...
Figure 4.2 Reversible transformations between the more hydrophobic SP state ...
Figure 4.3 Schematic diagram of a visible light stereolithography-based biop...
Figure 4.4 Microtomography (micro-CT) reconstructed images of the 3D-printed...
Figure 4.5 (a) Schematic representation of photocleavage process. (b) Molecu...
Figure 4.6 (a) Scheme (up) and stress–strain curves (down) of photodegradabl...
Figure 4.7 (a) Scheme of fabrication of SF@PDA composite porous structure an...
Chapter 5
Figure 5.1 Degradation of stimuli-responsive materials mediated by biologica...
Figure 5.2 Degradation of stimuli-responsive materials mediated by chemical ...
Figure 5.3 Degradation of stimuli-responsive materials mediated by physical ...
Figure 5.4
In vitro
cytotoxicity evaluation assays: (a) extract test, (b) di...
Chapter 6
Figure 6.1 Bioeffects induced by US stimulation. The ultrasonic stimulation ...
Figure 6.2 US-stimulated biomaterials for bone regeneration applications. US...
Figure 6.3 Magnetically assisted bone regeneration. The combination of
magne
...
Figure 6.4 A schematic of the direct piezoelectric effect, defined as voltag...
Figure 6.5 Schematic illustration showing the fabrication process and applic...
Chapter 7
Figure 7.1 Anatomic structure and organization of skeletal muscle tissue....
Figure 7.2 Heart myocytes’ excitation–contraction relationship.
Figure 7.3 An overview of the distinctive characteristics and cellular proce...
Figure 7.4 (A) Operation of the biomimetic contractile apparatus: (a) Human ...
Figure 7.5 Illustrative representation of the synthesis of multifunctional F...
Figure 7.6 Impact of magnetic scaffolds in conjunction with magnetic fields ...
Figure 7.7 (A) Diagrammatic representation of the fabrication of Gel-bFGF. (...
Chapter 9
Figure 9.1 Layers of the epidermis. The epidermis is composed of five layers...
Figure 9.2 Phases of wound healing.
Figure 9.3 Material requirements for wound healing.
Figure 9.4 The transepithelial potential difference in intact skin (left) is...
Chapter 10
Figure 10.1 Targeting and stimuli for responsive polymers in cancer treatmen...
Figure 10.2 Most common pH-dependent action mechanisms: (a) ionizable pH-sen...
Figure 10.3 Redox-responsive polymer.
Figure 10.4 Enzyme-responsive polymer.
Figure 10.5 (a) Coil-to-globule transition in thermo-responsive polymers. (b...
Figure 10.6 Magnetic-responsive polymers.
Figure 10.7 Neutron-responsive polymer for BNCT.
1
Figure 1 Representative examples of smart material types used for different ...
Guide
Cover
Table of Contents
Title Page
Copyright
Preface
Begin Reading
Summary and Outlook
Index
End User License Agreement
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Stimuli-Responsive Materials for Tissue Engineering
Edited by
Clarisse Ribeiro
Unai Silván
Senentxu Lanceros-Méndez
Editors
Dr. Clarisse RibeiroUniversidade do MinhoBraga 4710-057Portugal
Dr. Unai SilvánBasque Center for Materials, Applications and NanostructuresUPV/EHU Science ParkLeioa 48940Spain
Prof. Senentxu Lanceros-MéndezBasque Center for Materials, Applications and NanostructuresUPV/EHU Science ParkLeioa 48940Spain
Cover Images: © Romanova Natali/Shutterstock, © ThinkNeo/Getty Images, © Ed Reschke/Getty Images, © PansLaos/Getty Images
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Preface
“The world was to me a secret which I desired to divine.”
Mary Wollstonecraft Shelley (1797–1851);“Frankenstein; or, The Modern Prometheus” (1818)
One of the first stimuli-responsive materials, an alloy composed of gold and cadmium, was described in the year 1932. Since then, many new materials that change their properties in response to external triggers have been developed. It is now possible to design and synthesize materials that alter their electric surface charge, rigidity, shape, and degradation rate by exposing them to changes in temperature, light, humidity, magnetic fields, or mechanical stresses, among other possibilities. Further, two or more of these advanced properties can be combined in a single construct, expanding the versatility and applicability of the individual materials.
The emergence of these smart and multifunctional materials has the potential to transform a wide variety of technological areas, including tissue engineering. Classically, much of the research in this field has been aimed at exploiting the therapeutic potential of stem cells. However, the importance of creating a niche to provide cells with the necessary bioactive factors and physical stimuli has been recognized as central to a successful regeneration process. This microenvironment is composed of biochemical factors and a number of complementary physical stimuli, which vary depending on the specific organ and include, among others, mechanical forces, temperature variations, electrical signals, or combinations of these. For instance, tissues of the musculoskeletal system, which comprises bones, muscles, tendons, and joints, are exposed to intense mechanical loading. However, in bone, the mechanical loading is associated with shear stresses generated by interstitial fluid flow in the lacunar-canalicular network and surface potential changes due to the piezoelectric properties of the bone matrix, both of which are known to impact the activity of resident cells. This example provides an insight into the complexity of tissue homeostasis and highlights the importance of considering multiple factors in the design of suitable therapeutic strategies. In this context, the use of multifunctional smart materials with dynamically controlled properties represents a unique tool for the repair of damaged tissues. In addition, smart materials can be engineered to respond to specific environmental cues related to the progress of the healing process and trigger, for instance, the release of bioactive molecules or the adjustment of the material’s physicochemical properties, providing an adaptive microenvironment. Furthermore, the response of the material and the required triggering signals can be tuned based on individual characteristics, promoting customized approaches for tissue engineering and regenerative medicine. In addition to these applications for tissue and organ regeneration, other smart material-based approaches are conceived as a treatment for cancerous lesions. To this end, smart materials are generally used in the form of nanoparticles that release their antitumoral cargo either in response to external stimuli or to cancer-specific environmental cues, such as low pH or the presence of specific biomolecules.
In this monographic volume, we explore the transformative potential of smart and multifunctional materials in the field of tissue engineering. In the first part of the volume, we focus on materials with different responsive properties. Specifically, Chapters 1 and 2 are focused on electroactive and magnetoactive materials, respectively. In Chapter 3, shape-memory materials are introduced, and Chapter 4 is devoted to photo-responsive materials. In turn, Chapter 5 discusses materials for which their degradation rate can be controlled. The second part of the volume focuses on strategies that make use of stimuli-responsive smart materials for the regeneration of specific organs and tissues. Chapters 6 and 7 cover two tissues of the musculoskeletal system, bone and muscle, respectively. Chapter 8 deals with neural tissue, and Chapter 9 focuses on skin. Finally, Chapter 10 gives an overview of the potential of smart materials for the treatment of cancer.
In this way, the main concepts, strategies, and a comprehensive state of the art are provided both from the perspective of the potential of smart materials and from the point of view of tissue regeneration strategies. We are convinced that this book will provide the reader with up-to-date knowledge and deep insights in the field of advanced materials and biomedical sciences. We are also convinced that this book will serve to promote further research, development, and translation efforts in this fascinating and needed field.
We want to warmly thank the authors of the different chapters for their excellent contributions, allowing a broad view of the different areas with precise, concise, and well-elaborated information. Their expertise and excellent contributions in the field over the years are represented in this book in the most scientifically excellent and pedagogical way. It has been a real honor and a great pleasure to work with you on the relevant achievement that this book represents. Thanks a lot!
We would also like to express our gratitude for the support we have received over the years from our research groups and the research centers at the University of Minho, Braga, Portugal, and BCMaterials, Basque Center for Materials, Applications and Nanostructures, Leioa, Spain, to which the different editors belong. The great environment, deep discussions, dedication, understanding, and support we have experienced during this time have been a driving force in making possible the materialization of this book, which also contains a piece of each of you.
Further, this book could not have been possible without the excellence, kindness, and patience of the team at Wiley: Thanks a lot to Martin Preuß, Daniela Bez, and Monica Chandrasekar for their great and continuous support! It has been a real pleasure!
Finally, we are convinced that this book will become a milestone in this relevant field to further foster scientific, engineering, and medical efforts, leading advances in tissue regeneration strategies. These advances will allow tissue regeneration to become a widespread reality, with the consequent impact on improving the quality of life in many critical medical scenarios waiting for a proper solution.
We encourage researchers and professionals in the area, graduate and undergraduate students, and policymakers developing strategies in the area to take advantage of the information and scope provided in the present book to successfully tackle the relevant challenge of tissue regeneration.
BCMaterials, Leioa, Spain18 Februray 2024
Clarisse Ribeiro
Unai Silván
Senentxu Lanceros-Méndez
Part ISmart and Multifunctional Materials for Tissue Engineering
1Electroactive Materials for Tissue Engineering
Teresa Marques-Almeida1, Estela O. Carvalho1, Unai Silvan2,3, Senentxu Lanceros-Méndez1,2,3, and Clarisse Ribeiro1
1University of Minho, CF-UM-UP–Physics Centre of Minho and Porto Universities and LaPMET–Laboratory of Physics for Materials and Emergent Technologies, Campus de Gualtar, 4710-057 Braga, Portugal
2BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain
3Basque Foundation for Science, Ikerbasque, 48009 Bilbao, Spain
1.1 Introduction
Tissue engineering (TE) is a branch of regenerative medicine that aims to repair and restore damaged or lost tissues using biological substitutes [1]. TE combines three key factors to seek this ultimate goal: cells, scaffolds, and biochemical and/or biophysical cues [2]. Scaffolds are used to provide a supportive setting that encourages cell growth and matrix synthesis, thereby promoting the formation of new tissue. In turn, biochemical and biophysical cues are employed to create the optimal microenvironment for long-term communications between cells and surrounding tissues/organs. The ultimate objective is to closely mimic the native microenvironment of the impaired tissue [2, 3].
Besides the well-established biochemical cues provided by bioactive factors, electrical signals play a crucial role in cell activity as a biophysical cue. Electrical signals regulate a variety of typical physiological processes, from brain activity to heartbeat [2, 4]. In this context, electroactive biomaterials have emerged as one of the most promising scaffolds for TE application in recent decades. These materials promote conduction of electrical charges and exchange of ions with the surrounding environment. Therefore, the incorporation of electroactive materials into scaffolds for TE can provide a platform for delivering electrical signals to cells, promoting cell proliferation or differentiation, and consequently tissue regeneration [5]. The characteristics of these materials can be modified by adjusting their composition, morphology, and processing conditions to optimize their performance for specific TE applications.
An in-depth understanding of how the cellular microenvironment evolves over time and the pathways that might be used to impose electricity on cells/tissues via electroactive materials is critical for the development of active scaffolds. Additional investigations in this field aim to develop tunable electroactive materials with enhanced biocompatibility and improved electrical properties, opening up new possibilities for TE and regenerative medicine [5, 6].
1.2 Relevance of the Electrical Signals in the Human Body
Human body functions and homeostasis are influenced by physical stimuli [7]. Electromagnetic radiation, temperature, and mechanical forces are external physical stimuli known to present a significant impact on biological events. On the other hand, intrinsic electrical signals and mechanical forces (compressive loading, hydraulic pressure, shear stress, and tensile forces) are the internal physical stimuli that also have been demonstrated to naturally control cell fate by enhancing cell target functions such as adhesion, migration, proliferation, and differentiation [8]. Although all physical stimuli are important and have relevance on the cellular level, it is widely accepted that electrical signals are the most prominent physical stimuli for controlling many physiological processes and may even outperform other physical cues [2, 4, 9].
In the eighteenth century, Luigi Galvani and coworkers demonstrated the presence of an intrinsic form of electricity responsible for nerve conduction and muscle contraction. For the first time, they reported the possibility of causing muscle twitches in freshly killed animals using electric signals and then demonstrated the existence of the injury potential [10]. Since then, many researchers corroborated those findings and clearly defined phenomena such as membrane potentials (Vm) and later action and resting potentials. Currently, it is stablished that Vm are fundamental features of all cells and are caused by the movement of ions across the cell membrane, which results in charge separation and the generation of an electrical potential difference. This occurrence is both a by-product and a regulator of a wide range of essential properties at multiple biological organization levels, intrinsic to the normal function of all cells, organelles, and molecules [9, 11]. For instance, these signals drive processes such as respiration, influence pH levels, and modulate the redox state. Additionally, they facilitate cell-to-cell communication, guide cell migration (in a process known as galvanotaxis), and contribute to tissue repair [12].
Electrical signals enable cells to communicate with each other at the tissue and organ levels, which in turn plays a crucial role in the organ’s performance [13]. Various types of tissues exhibit specific electrically conductive and/or electrically responsive properties, such as piezoelectricity and ferroelectricity, and thus several functions are controlled by electrical signals. Figure 1.1 depicts tissues that inherently make use of these signals to regulate different physiological processes [2, 4], such as neural communication [14, 15], bone regeneration [16, 17], heartbeat activities [18, 19], muscle contraction [20, 21], and wound healing [22, 23]. Moreover, compelling evidence indicates that alterations in cellular and tissue excitability, along with more extensive electrical fields across tissues, may have implications for embryo development and tumorigenesis [12, 24].
Figure 1.1 Electric activity in the human body.
All these organs and tissues are known as electroactive tissues given their ability to generate and transmit electrical signals [13]. The nervous system exhibits the highest electrical activity, facilitating the transmission of signals from neurons through synapses to their respective destinations. The pacemaker cells in the heart, a specialized subpopulation of cardiomyocytes, generate rhythmic impulses that propagate through the entire heart and trigger mechanical activity, i.e., heart beating, in ventricular myocytes [6]. Bone exhibits piezoelectric properties, and it generates electrical charges in response to mechanical stress, which ultimately triggers cell growth and repair. Regarding skin, the normal electrical fields are disrupted after an injury, resulting in abnormalities. The Vm is severely disrupted, and a wound electrical field forms, driving cells to the wound for healing purposes, such as epithelial cells in skin injuries [25].
Given the importance of electrical cues in physiological tissue function, disease manifestation and progression, and regeneration, extensive research has been conducted to identify the ideal conditions for electrically stimulating tissues and modulating cell response in vitro, in order to understand the action mechanisms and develop functional therapeutic interventions, especially in the scope of TE. Table 1.1 provides a few examples of the influence of in vitro and in vivo electrical stimulation on different mammalian cell types. Notwithstanding, the given stimulation may vary depending on several factors, such as the strength and frequency of the electric field, its duration, and specific characteristics of the cells being stimulated.
Table 1.1 Overview of in vitro and in vivo electrical stimulation effects on different mammalian cells.
Cell type
In vitro
effect of electrical stimulation
In vivo
effect of electric stimulation
Refs.
Neurons
Enhanced neurite outgrowth, improved myelinization, and increased synaptic activity
Enhanced axonal sprouting, and improved functional recovery after spinal cord injury
[
14
,
15
,
26
,
27
]
Skeletal muscle cells
Enhanced myogenic differentiation and myotubes contraction
Improved muscle function in mice with muscular atrophy
[
20
,
21
,
28
,
29
]
Bone cells
Increased osteoblast cell proliferation, suppressed osteoclast recruitment, and enhanced calcification
Improved bone regeneration, and increased matrix formation around orthopedic implants
[
16
,
30
,
31
]
Skin cells
Increased collagen production, stimulation of proliferation, and differentiation of keratinocytes, fibroblasts, and endothelial cells
Enhanced epithelialization and improved wound healing
[32]
Cardiac muscle cells
Enhanced stem cell differentiation, improved contractile function and maturation, and promoted cardiomyocyte alignment and synchronization
Promoted angiogenesis, reduced apoptosis, and inflammation in ischemic myocardium
[
33
–
35
]
Cancer cells
Reduced proliferation, apoptosis induction, and metastasis suppression
Enhanced sensitivity to usual therapies, and decreased tumor growth in mice with breast cancer
[
36
,
37
]
The evaluation of electric fields in a biological context has led to a better understanding of several aspects of living organisms, including their pivotal role in tissue development, regeneration, and repair processes. Furthermore, it has shed light on the mechanisms underlying cellular detection of electric fields and subsequent modulation of downstream signaling pathways, responsible for orchestrating cellular responses. These discoveries have not only opened up new avenues for scientific inquiry but also hold great promise for the development of innovative therapeutic interventions [38].
1.3 Relevance of the Electrical Signals in Cell Processes
The delivery of electrical signals to cells is closely related to changes in their Vm. Every cell presents a Vm that reflects the electrical potential difference across its plasma membrane. This potential is determined by the balance of ionic concentration on both sides of the membrane, and this balance is tightly regulated by ion channels and pumps [39].
Almost all cells present a Vm in the range of −10 to −90 mV at a steady state [40]. However, the specific value varies among different cell types, for instance, neurons typically exhibit a Vm around −70 mV [41], while cardiac cells tend to be closer to −90 mV [42]. The maintenance of this long-term steady state is critical for regulating cell proliferation and establishing tissue-level behaviors and patterns. Nevertheless, this state is influenced by several factors, including a diverse array of ion channels, variations in the expression of channels and isoforms with distinct response characteristics and ion affinities, and posttranslational channel alteration [40].
The difference in the electrical potential across the cell membrane is dynamic in the vast majority of cells. The mechanism through which each cell manages electrical signals determines whether the cell is excitable or non-excitable [43]. In excitable cells, such as muscle and neuron cells, Vm is also named resting potential and defines the non-excited state of the cell responsible for internal processes such as basal metabolic activities that sustain their homeostasis and prepare for forthcoming signaling events. These cells can change to non-resting states, known as action potentials, which correspond to the moment when the cell is actively involved in signaling, communication, or other forms of cellular activity [44]. Action potentials involve a rapid change in Vm when excitable cells receive a stimulus that exceeds a certain threshold. The stimulus triggers a chain of events that includes the opening and closure of voltage-gated ion channels, resulting in a distinct shift in Vm, as represented in Figure 1.2. In the nervous system, it serves the purpose of transmitting signals throughout the body. In skeletal and cardiac muscle, action potentials are responsible for coordinating and regulating the excitation–contraction coupling. Despite not having an action potential, non-excitable cells frequently have a dynamic Vm (Figure 1.2) [44], which serves a wide range of essential biological functions [14, 16, 45, 46]. Additionally, in excitable cells, depolarization triggers the opening of voltage-dependent Ca2+ channels (VDCC), resulting in calcium influx. Ca2+-activated K+ (KCa) channels, particularly large-conductance Ca2+-activated K+ channels (BKCa – Big potassium calcium-activated channels), are activated and subsequently induce hyperpolarization, thereby reducing Ca2+ influx through VDCC. Thus, BKCa channels act as a negative feedback mechanism to VDCC activity. In non-excitable cells, Ca2+ channels mainly consist of voltage-independent calcium channels (VICC) instead of VDCC. Ca2+ release-activated Ca2+ (CRAC) channels (VICC type) activate KCa channels, leading to hyperpolarization [47]. Unlike VDCC, CRAC channels are not closed by hyperpolarization since they lack a voltage-sensing domain. Stromal interaction molecules open the pores of CRAC channels, enabling them to conduct Ca2+ even at hyperpolarized potentials. The hyperpolarization induced by the KCa channel increases the driving force for Ca2+, promoting its influx through the CRAC channel. Consequently, BKCa channels contribute to positive feedback for Ca2+ influx through CRAC channels. Activation of CRAC channels results in minimal membrane depolarization due to their lower single-channel conductance compared to VDCC. The slight depolarization resulting from Ca2+ influx through CRAC channels is counteracted by the significant membrane hyperpolarization caused by K+ conductance through the BKCa channels [48, 49].
Figure 1.2 Correlation between changes in Vm and intracellular calcium concentration ([Ca2+]) in both (a) excitable and (b) non-excitable cells.
Calcium influx is a fundamental requirement for normal matrix metabolic and secretory functions [50, 51], the change in Vm being closely related to calcium influx, independently of the cell type [37]. It can boost cell metabolism and promote adenosine triphosphate (ATP) depletion, resulting in cytoskeleton reorganization and alterations in membrane-related cellular activities like endocytosis, exocytosis, adhesion, migration, and proliferation [52].
Although the Ca2+ release-activated Ca2+ (CRAC) channel is primarily responsible for calcium influx in non-excitable cells (Figure 1.2b), voltage-gated ion channels are also expressed in these cells and are also responsible for cell membrane depolarization, similar to what happens in excitable cells [53–55]. The precise physiological functions assigned to these channels in non-excitable cells, as well as their regulation mechanisms, are still debated and being researched [56, 57]. However, it is well established that electrocoupling is the response mechanism to electrical stimulation in the widest range of cells, both excitable and non-excitable cells, such as neurons [58], pancreatic and bone cells, and even tumor cells [54]. Exposing cells to electric fields can directly activate L-type voltage-gated calcium channels, a type of VDCC. This activation triggers several regulatory responses through enzymatic activities, enhancing the expression of differentiation-related genes [59]. An example of a molecular signaling pathway that triggers cell differentiation processes in the presence of electrical fields is the cyclic adenosine monophosphate (cAMP)-dependent pathway [58, 60, 61]. This pathway is activated by the elevated intracellular concentration of Ca2+, which leads to the activation of adenyl cyclase (AC). The AC catalyzes the conversion of ATP to cyclic AMP (cAMP), a secondary messenger molecule involved in numerous regulatory signaling pathways [62]. Thus, the activation of VDCC by electrical pulses increases cellular Ca2+ influx and consequently starts the AC signaling cascade. In addition to this pathway, the pERK and Wnt/β-catenin pathways are also activated by the opening of the VDCC and play an important role in stem cell differentiation [63, 64]. Note that calcium oscillations were always the trigger [54, 59, 62, 65].
Besides calcium ions, additional electrical-signal-related pathways regulate proliferation, differentiation, and apoptosis [37, 55]. Figure 1.3 summarizes possible pathways involved in biological response to electrical stimulation.
Reactive oxygen species (ROS) are thought to be important mechanisms involved in cell response to electric signals. Controlled induction of ROS at physiological levels can improve interactions with signaling molecules [66]. Several studies have demonstrated that moderate ROS levels activate mitogen-activated protein kinase (MAPK) cascades, which are central signaling pathways that regulate cellular processes, including proliferation, differentiation, and apoptosis. Thus, a mild increase in hypoxia-induced ROS has been revealed to mediate cell proliferation and differentiation [67–69].
Regarding transmembrane proteins, it has been reported that electrical signals can reorganize the membrane proteins and lipids on the cell’s external site due to the induction of a relative electrophoretic movement of these components on the cell’s exterior [70]. Electrical fields can up-regulate the expression of epidermal growth factor receptor (EGFR) on fibroblasts, corneal epithelial cells, and keratinocytes [69], inducing its redistribution and accumulation primarily on the cathode side of the cell. Besides this asymmetric distribution, it can cause the colocalization of membrane lipids, resulting again in the triggering of MAPK signaling cascades [69, 71].
Figure 1.3 Possible intracellular responses to electrical stimulation. Membrane receptors, ion channels, integrins, and gap junctions mediate electrical stimulation, triggering responses such as cell proliferation, migration, and differentiation.
Source: Liu et al. [40]/John Wiley & Sons.
Considering the previous remarks, it appears that the application of electrical stimuli in diverse tissues, whether excitable or non-excitable, might be an efficient strategy for repairing injured tissues due to their ability to promote tissue proliferation and differentiation. Even though each tissue has a distinct response to electrical signals with varying degrees of sensitivity, electrical signals appear to trigger biochemical and physiological processes, leading to effective and specific tissue regeneration responses. By applying controlled electrical stimulation, electroactive materials can harness this potential and actively influence tissue repair. They offer a unique and powerful approach to tissue regeneration by delivering electrical stimulation that mimics natural cellular microenvironments.
1.4 Types of Electroactive Materials
Electroactive materials are a subset of smart materials that can translate a physical or chemical stimulus into an electrical signal, and vice versa, in a reproducible and predictable manner. They are used in a wide range of applications, including sensors, actuators, energy-harvesting, and biomedical devices [72]. Further, electrically conductive materials also play a relevant role in the area of tissue engineering and will be considered in this section. As the basic principle of tissue engineering is to mimic the extracellular microenvironment and, as described, electrical signals are one of the human body’s main physical stimuli, electroactive materials and electrically conductive materials are being highly pursued for tissue engineering purposes. Among the different electroactive materials, in the following, conductive, piezoelectric, magnetoelectric, and thermoelectric materials will be considered based on their relevance in the tissue regeneration area.
1.4.1 Conductive Materials
Conductive materials can conduct electricity through them, presenting low resistance to the movement of electric current, whether the charge carriers are ions or electrons [73]. The conductivity (σ, S/m) of a material represents its ability to conduct electric current and can be quantified by Eq. (1.1), where R is the material’s electrical resistance, A is its cross-sectional area, and l is its length.
The electronic structure of a material defines whether a material is an insulator, a semiconductor, or a conductor. The band gap theory is commonly used to distinguish these three types of materials (Figure 1.4a). Conductors are characterized by valence bands overlapping the conduction bands, allowing the valence electrons to move freely and propagate in the conduction band. Semiconductors have small energy gaps and, upon excitation, electrons can cross to reach the valence band, allowing the conduction of current. On the other hand, insulators have larger band gaps that cannot be crossed by electrons and thus disabled them from conducting current [74]. As electrons in polymers are not delocalized, they cannot flow easily from atom to atom to conduct currents. Hence, conductive polymers’ conductivity cannot be explained by the gap band theory; rather, it is explained by the existence of a conjugated backbone, whereby carbon atoms are alternately connected by single and double bonds (Figure 1.4b). The single bond detains a strong localized s-bond, and the double bond has a weaker localized p-bond and a stronger s-bond. This creates a continuous overlapping of pz-orbitals in the chain of p-bonds allowing for p-bonds electrons to be easily delocalized and transferred along the polymer chain, consenting electrical flow [46, 74].
Figure 1.4 (a) Band gap theory of conductors, semiconductors, and insulators; (b) Illustration of a conductive polymer-conjugated backbone with alternating patterns of double and single bonds.
Electronic conductors include metals (e.g., gold, silver, iron), members of the carbon family (e.g., carbon nanotubes (CNTs) and graphene), and conductive polymers (CPs), while among ionic conductors are polymer electrolytes (e.g., poly(vinyl alcohol) (PVA) and chitosan), solid-state ionic conductors (e.g., polyethylene oxide (PEO)), and ionic liquids (ILs). Due to their versatility, conductive materials can be designed to meet specific requirements concerning the target tissue. Metal-based nanoparticles, CNTs, graphene-based materials, and ILs are being used for TE applications [3]. They can be used purely or in the form of composites with other materials and have shown suitable results in promoting cellular activities, ranging from facilitating electrical signaling in neurons to influencing cell migration, adhesion, proliferation, and differentiation in different cell types [75–78]. However, the biological application of inorganic materials is limited, either by the cost or by cytotoxicity problems [46]. Organic CPs allow the combination of good biocompatibility, and chemical and mechanical properties of polymers, with the electrical and optical properties of metals, which makes them highly pursued to overcome the drawbacks of inorganic materials [79]. The first CP discovered was polyacetylene (PA) in 1977, leading to the Nobel Prize in Chemistry for Alan MacDiarmid, Alan Heeger, and Hideki Shirakawa in 2000. Since then, a variety of CPs has been investigated, including polypyrrole (PPy), polyaniline (PANI), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(p-phenylene vinylene) (PPV), and polyacetylene (PAc) [80]. CPs have lower conductivity compared to most metals; thus, in order to increase their conductivity or improve performance, CPs are often doped or blended with other materials, and can reach electrical conductivities as high as 102–106 S/m [79, 81, 82].
CPs’ electrical conductivities range meets the conductivities present in many biological tissues, making them valuable materials for creating biomimetic environments that can influence and support cellular functions [83]. PPy, PANI, and PEDOT are the most used for TE and are particularly interesting for neural and cardiac excitable cells, and also for non-excitable bone cells [84–86]. Non-excitable cells/tissues can generate endogenous charges that stimulate regenerative responses through VDCCs activation, as it is the case of bone cells [85]. The frequent application of CPs in this field arises from the suggestion that bone healing can be promoted by restoring the bioelectrical microenvironment [87]. An overview of the most relevant conductive materials for TE applications is presented in Table 1.2.
The main drawback of conductive materials is the need for invasive electrodes, which can be overcome by piezoelectric or magnetoelectric materials, that can be remotely stimulated.
1.4.2 Piezoelectric Materials
Piezoelectric materials are the class of electroactive materials that can translate mechanical inputs into an electric output (direct piezoelectric effect), or, alternately, generate a mechanical output when exposed to an electric field (inverse piezoelectric effect).
The direct piezoelectric effect was first discovered by the Curie brothers, Jacques and Pierre Curie, in 1880 [106], and the inverse piezoelectric effect was mathematically deduced by Lippman in 1881 [107] and later confirmed by the Curie brothers. The piezoelectric effect, or piezoelectricity, occurs because of the crystal structure of these materials that lacks a center of symmetry (illustrated by the quartz piezoelectric effect in Figure 1.5).
Table 1.2 Overview of relevant conductive materials for TE applications.
Conductivity (S/m)
TE applications
Inorganic conductive materials
Gold
4.9 × 10
7
[88]
Bone [
89
–
91
], muscle [
92
,
93
], skin [
94
,
95
], neuro [
96
–
99
], cardiac [
100
–
102
]
CNTs
10
6
–10
7
[103]
Graphene
10
8
[103]
Organic conductive materials
PPy
10
2
–10
5
[104]
Bone [
84
,
90
,
105
], muscle [
84
,
92
], skin [
45
,
84
,
105
], neuro [
84
,
96
,
97
,
105
], cardiac [
84
,
101
,
105
]
PANI
10
4
[82]
PEDOT
10
4
–10
5
[104]
Figure 1.5 Piezoelectric effect in quartz.
This non-centrosymmetric structure gives rise to a non-centrosymmetric arrangement of positive and negative ions, resulting in dipolar moments. When a mechanical stress is applied, it disrupts the dipole moments, causing a charge redistribution across the two faces of the crystal lattice, resulting in the generation of an electrical potential proportional to the applied force. Conversely, when an electric field is applied, the dipolar moments within the crystal structure suffer variations along the electric field direction, and the material suffers a mechanical deformation proportional to the applied electrical field. The piezoelectric effect can be quantified using Eq. (1.2) for direct piezoelectric coefficient (dij, pC/N), or Eq. (1.3) for inverse piezoelectric coefficient (eij, C/m2), where D is the electric displacement field, E corresponds to the electric field strength, and X and x are the mechanical stress and strain, respectively [108].
Piezoelectricity is found in many biological tissues, including bone, skin, tendon, and cartilage, and has been referred to as an extensive and fundamental feature of biological tissues since it is implicated in essential physiological events [109]. As TE is mainly based on biomimetic approaches, the interest and research on these electroactive materials have been strongly increasing [110]. Piezoelectric materials can generate local electrical potentials with non-invasive methods, without the need to use electrodes and/or wires that could lead to tissue inflammation when implanted, and instead using vibration plates, sound, or ultrasound (US) stimulation, among others [41].
Piezoelectric materials can be inorganic, organic, or combination of them in composites. Among inorganic are quartz, lead zirconate titanate (PZT), barium titanate (BaTiO3), and zinc oxide (ZO); and among organic are synthetic polymers such as nylon, poly(vinylidene fluoride) (PVDF), PVA, poly(urethane) (PU), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)), and natural polymers including chitosan, silk, collagen, and cellulose. Although inorganic piezoelectric materials have higher piezoelectric response, they can be fragile and show limited or no biocompatibility. This makes organic piezoelectric polymers the most researched for TE applications, due to their biocompatibility and tunable mechanical characteristics [109, 111]. Further, they are easier to process in a wide variety of morphologies [110].
Table 1.3 Overview of relevant piezoelectric materials for TE applications.
Piezoelectric coefficients (
d
ij
, pC/N)
TE applications
Inorganic piezoelectric materials
PZT
225–590
[115]
Bone [
116
,
117
], muscle
[118]
, skin
[119]
, neuro
[116]
, cardiac [
19
,
116
,
117
], muscle
[118]
, skin
[119]
, neuro
[116]
, cardiac
[19]
BaTiO
3
191
[115]
ZnO
13
[115]
Organic piezoelectric materials
PVDF
24–34
[109]
Bone [
120
,
121
], muscle
[121]
, skin
[122]
, neuro
[41]
, cardiac [
19
,
120
,
121
], muscle
[121]
, skin
[122]
, neuro
[41]
, cardiac
[19]
P(VDF-TrFE)
38
[109]
PLLA
9.82
[109]
PHBV
1.3
[123]
Silk
1.5
[124]
PVDF and its copolymers, as poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)), are the most explored for TE applications due to their high dielectric constants (6–12) and piezoelectric coefficients (|d33| = 24–34 pC/N) [109]. In the last decade, electrical stimulation delivered by PVDF-based scaffolds has been strongly focused on bone TE applications [17, 31, 112] and expanded to other tissues, such as muscle [21] and nerve [113, 114]. PLLA, PHBV, silk, cellulose, and collagen are also being investigated because of their biodegradability, although they present lower piezoelectric coefficients. An overview of the most relevant piezoelectric materials for TE applications is presented in Table 1.3.
1.4.3 Magnetoelectric Materials
Most magnetoelectric materials applied in the area of tissue regeneration are composite materials that integrate magnetic and piezoelectric components. The coupling of these materials properties results in a magnetoelectric coupling, which allows a magnetic field to switch/tune the polarization state of the material (direct magnetoelectric effect), and, conversely, its magnetization can be switched/tuned by an electric field (inverse magnetoelectric effect) [125, 126]. The magnetic component of the magnetoelectric system, which can have magnetic and magnetostrictive properties, can sense an applied magnetic field and translate it into a mechanical deformation, that is then converted by the piezoelectric component into an electrical potential (Figure 1.6) [127].
Figure 1.6 Magnetoelectric effect using (a) magnetostrictive particles or (b) magnetic particles.
Source: Ribeiro et al. [127]/with permission of Elsevier.
Magnetoelectricity was first described by Pierre Curie in 1894 [128] and experimentally observed by Astrov in 1960 [129]. Magnetoelectric scaffolds are a relatively new TE paradigm. Magnetic nanoparticles such as iron oxides (Fe3O4, Fe2O4), iron-based metal oxides (CoFe2O4), and Terfenol-D are frequently combined with piezoelectric polymers such as PVDF, P(VDF-TrFE), PLLA, PLGA, and PHBV [111,130–135]. Magnetic fields have been implemented for a wide range of biomedical applications, from magnetic resonance, and target drug delivery to hyperthermia, due to their deep tissue penetration and highly controllable motion of magnetic materials [40]. Thus, the use of magnetoelectric scaffolds may be effective in cases where the patient is immobilized and natural mechanical stimulation is not fully guaranteed, allowing the deployment of an external magnetic field to remotely promote tissue recovery [130]. These types of electroactive scaffolds are being highly investigated for bone and nerve regeneration [111,135–138]. Since magnetic fields can penetrate brain tissue with limited signal attenuation and side effects, they are pursued from deep brain stimulation in neural tissue engineering applications [136].
1.4.4 Thermoelectric Materials
Thermoelectric materials are another class of smart electroactive materials, characterized by the ability to convert heat into electrical energy, or vice versa [139]. The thermoelectric effect applied in biomedicine is mainly based on the Seebeck effect, which was discovered in 1821 by Thomas Seebeck and consists in the generation of an electrical voltage by a temperature difference between two materials or regions [140]. When a temperature difference is applied across a material made of two metals or semiconductors – one n-type and one p-type – induces the movement of charges leading to the generation of a voltage difference (Figure 1.7a). Later in 1834, Jean-Charles-Athanase Peltier discovered the Peltier effect, which is essentially the reverse of the Seebeck effect as it generates a temperature variation from the application of an electrical current. When an electrical current is applied across two metals or semiconductors, heat is either absorbed or released, depending on the direction of the current (Figure 1.7b). The thermoelectric effect comprises a third less-applied effect, called the Thomson effect. Discovered by William Thomson (later known as Lord Kelvin) in 1854, it establishes a mathematical relationship between the Seebeck and Peltier coefficients. It is distinguished by the generation of a temperature difference within a single conductor material when an electric current flows through it [141].
Figure 1.7 Thermoelectric materials: (a) Seebeck and (b) Peltier effects.
The performance of thermoelectric materials is measured by the dimensionless figure of merit (ZT), quantified by the Eq. (1.4), where S is the Seebeck coefficient, σis the electrical conductivity, and K is the thermal conductivity. Typically, thermoelectric materials have low thermal conductivity and high electrical conductivity [142].
Thermoelectric materials can be inorganic, including bismuth telluride (Bi2Te3), lead telluride (PbTe), silicon-germanium (SiGe), and skutterudite (CoSb3); carbonaceous such as CNTs, and graphene; or organic, among them poly(3-hexylthiophene) (P3HT), PPy, PANI, PEDOT, and polythiophene (PTh) [139]. Inorganic thermoelectric materials are typically characterized by low mechanical performance, as they are brittle, are often toxic, and demand expensive microfabrication techniques for processing. Thus, organic polymeric thermoelectric materials are the most interesting alternatives for applications in the area of biomedicine, due to their low thermal conductivity, good flexibility, low cost, and non-toxicity [139, 143].
Thermoelectric materials and devices are being used in power generation, solid-state cooling, and heating. Regarding the biomedical field, these materials are mostly used to integrate biomedical sensor systems such as thermoelectric power generators (TEGs) or thermoelectrical coolers (TECs) [144]. Much research is being devoted for developing implantable and wearable TEGs, to assist specific human tissues or organs as implantable medical devices [145, 146].
1.5 Relevance of the Material’s Architecture
In addition to material and active response selection, it is crucial that the scaffold provides morphological cues resembling the tissue-specific microenvironment. The extracellular matrix of a defective site must be mimicked, with the scaffold providing cell support to fine tuning specific functions, as well as spatial and temporal guidance for multicellular processes of formation and regeneration of damaged or lost tissues. Therefore, the morphological features of the scaffold should meet a biomimetic approach, and thus be appropriate for the specific microenvironment in which it will be inserted.
Scaffolds with electroactive properties, particularly polymers or polymer-based materials, can be obtained in the most varied morphologies (Figure 1.8), including films, fibers, membranes, hydrogels, 3D structures, microspheres, or patterned surfaces, among others [147]. For their processing, and depending on the morphology requirements, there are several methods and techniques that can be used including doctor blade, spin-coating, screen printing, electrospinning, melt electrowriting, electrospray, phase separation techniques (e.g., thermally induced phase separation (TIPS), nonsolvent-induced phase separation (NIPS), and vapor-induced phase separation (VIPS)), salt leaching, soft lithography techniques (e.g., replica molding, microcontact printing, microtransfer printing, and micromolding in capillaries), and 3D printing, among others.
1.5.1 Films
For TE applications, films are often chosen for initial studies on the influence of material properties other than their morphology, such as the electroactive response, as they provide a simple flat surface that resembles conventional 2D culture conditions. Films can be processed by several techniques that include doctor blade and spin-coating, and printing technologies such as screen printing, and spray printing. Doctor blade, also known as blade or knife coating, is a widely used technique to obtain thin films on large area surfaces and is the most used for TE applications. The gap size between the blade and the substrate, and the polymer mass fraction define the thickness of the film. The blade is moved across a flat substrate in lab-scale production, whereas the substrate is moved, or the blade can be fixed for large-scale processes [147, 148]. After solvent evaporation, the final thickness of the film (d) decreases and is dependent on several parameters, such as the flow behavior, speed of coating, substrate surface energy, surface temperature, fluid surface tension, and viscosity, being determined by Eq. (1.5), where w is the gap width, c the concentration (g/cm3), and ρ the density of the material in the film (g/cm3) [147].
Figure 1.8 Representative SEM images of the distinct structures/morphologies: (a) β-PVDF film obtained by the doctor blade technique after mechanical stretching. Inset: The α-PVDF film before mechanical stretching. Scale bars: 5 μm (main) and 10 μm (inset). (b) β-PVDF film obtained by spin coating. Scale bar: 5 μm. (c) P(VDF-TrFE) film obtained by screen printing. Scale bar: 10 μm. (d) Porous P(VDF-HFP) film obtained by NIPS. Inset: Cross-section. Scale bars: 10 μm (main) and 100 μm (inset). (e) Porous P(VDF-CTFE) film obtained by NIPS. Inset: Cross-section. Scale bars: 30 μm (main) and 100 μm (inset). (f) Porous β-PVDF film obtained by NIPS (cross-section). Scale bar: 100 μm. (g) Porous β-PVDF film obtained by TIPS. Scale bar: 100 μm. (h) β-PVDF scaffolds obtained by solvent-casting particulate leaching. Scale bar: 500 μm. (i) β-PVDF scaffolds obtained by solvent casting and 3D nylon template. Scale bar: 400 μm. (j) β-PVDF scaffolds obtained by freeze extraction with a 3D PVA template (optical microscope image). Scale bar: 1 mm. (k) Patterned porous P(VDF-TrFE) structure obtained by replica molding. Inset: Cross-section. Scale bars: 500 μm. (l) Randomly electrospun β-PVDF fibers obtained by electrospinning. Inset: The oriented electrospun PVDF fibers. Scale bars: 10 μm. (m) β-PVDF spheres obtained by electrospraying. Scale bar: 5 μm.
Source: Ribeiro et al. [147]/with permission of Springer Nature.
Surface charge and charge variation of piezoelectric and magnetoelectric biomaterials in bone, muscle, and nerve regeneration have been studied using scaffolds in film morphology [21, 112, 113, 132, 149, 150]. Besides, films with electroactive properties are designed for wound dressings due to their ability to adhere to the skin and protect the wound, as well as to be combined with different pharmaceutical compounds, including antibiotics, to promote wound regeneration and prevent bacterial infections [151–153].
1.5.2 Electrospun fibers
The fibrillar architecture of some extracellular matrix components, such as laminin, fibronectin, collagen, and elastin, has inspired the design of scaffolds with similar structures. Fiber scaffolds can be obtained by different methods and techniques, including phase separation, self-assembly, electrospinning, and melt electrowriting [154]. Electrospinning is the most used technique, due to its easy handling, low cost, and versatility. Through this technique, natural, synthetic, or composite polymer-based fibers can be obtained in random and oriented morphologies, with or without internal porosity, and with the most varied range of diameters, from micro- to nanofibers [155]. Because of fibers’ easy processing and tailoring and large surface area-to-volume ratio, electrospun fibers are one of the most required and studied morphologies for TE applications. The electrospinning setup is composed of a syringe pump, a syringe filled with a polymeric solution and connected to a metallic needle, a metallic collector, and a power supply with high-voltage directly connected to the needle and the collector. Its functioning is based on an applied voltage to the polymeric solution, which is expelled as an electrically charged viscoelastic jet, with a controlled flow rate given by the syringe pump, toward the metallic collector. During this trajectory, the solvent evaporates from the jet, and the polymeric fibers are deposited onto the collector. Besides solution formulation, electrospinning parameters can be optimized to tailor the obtained fibers, such as the flow rate, applied voltage, needle diameter, distance between the needle and the collector, and the type of collector used [147]. The use of a static collector allows to obtain fibers mats with random fiber distribution, and a rotative collector allows for obtaining aligned fiber morphologies.
Fibers not only mimic fibrous components of ECM but also enable the control of cell directionality by contact orientation, which is essential for specific applications. For instance, oriented fibers can be used to promote the directional growth of different cell types, such as neural [156, 157] and muscle cells [149, 158], to develop nerve guidance conduits to repair nerve defects [159, 160], and for the guidance of new bone formation [161, 162]. Electroactive fibers, either conductive, piezoelectric, or magnetoelectric composites, can deliver morphological and biophysical cues that are particularly interesting for TE [6, 163]. For instance, electrical stimuli given by fibrous scaffolds have proven to enhance cardiogenesis [164], myogenesis [165], neurite extension [166], and peripheral nerve repair [167].
1.5.3 3D Porous Scaffolds
The scaffold’s internal structure, such as porosity, pore size, and interconnectivity, is highly influential in cell development. These features enable cell infiltration, migration, and interconnection, nutrients and waste diffusion, and help with tissue vascularization [168]. Porous architectures can be reproduced into films, fibers, membranes, or more complex 3D structures, using a variety of techniques, including phase separation, gas forming, salt leaching, and solvent-casting on 3D templates [147]. The porosity of the scaffolds is intimately related to its mechanical performance, for instance, higher porosities, in most cases, lead to decreased Young’s modulus, but on the other side lead to an improved surface area that is available for cell attachment, so once again, the technique to be chosen is highly dependent on the application [169]. Porous scaffolds are often used for bone TE due to their resemblance with the trabecular bone [170, 171], but are also studied for other tissues such as neuronal [170, 171], muscle [172], and cardiovascular [173].
Because cells are surrounded by complex 3D microenvironments, porous 3D scaffolds are a relevant morphology for TE approaches [174]. These 3D structures can be obtained using the previously mentioned conventional methods but due to the scaffolds’ complex 3D structure, these methods can be disadvantageous in terms of causing cytotoxicity, because the solvents are more difficult to completely evaporate during processing, and scaffolds’ microstructure and resolution are more difficult to control [175]. As an alternative, rapid prototyping approaches can be used to develop 3D scaffolds. Rapid prototyping technologies, or additive manufacturing, include selective laser sintering, fused deposition molding, stereolithography, and 3D printing. These technologies are integrated with computer-aided design (CAD
