Nanotechnology in Regenerative Medicine and Drug Delivery Therapy [1 ed.] 9811553858, 9789811553851

Table of contents :
Acknowledgments
Contents
Abbreviation 1
Abbreviation 2
Chapter 1: Magnetically Actuated Scaffolds to Enhance Tissue Regeneration
1.1 Introduction
1.2 Biological Effects of Magnetic Fields on Tissue Regeneration
1.3 Biological Effects of Magnetic Materials on Tissue Regeneration
1.4 Combination of Magnetic Scaffolds and Magnetic Fields to Accelerate Tissue Regeneration
1.4.1 Bone
1.4.2 Cartilage
1.4.3 Endothelial, Cardiac, and Neuron Cells
1.4.4 Immune Cells and Supportive Cells
1.4.5 Mechanisms and Modulation of Mechanical Stimulations by Magnetic Materials and Magnetic Fields
1.5 Summary and Perspective
References
Chapter 2: Conductive Nanostructured Scaffolds for Guiding Tissue Regeneration
2.1 Introduction
2.2 Classification and Fabrication of Conductive Scaffolds
2.2.1 Conductive Polymers
2.2.2 Conductive Inorganic Nanoparticles
2.2.3 Summarization of Physical Properties for Conductive Scaffolds
2.3 Conductive Scaffolds in Myocardial Tissue Regeneration
2.3.1 Composite Scaffolds Containing Conductive Polymers
2.3.1.1 Polypyrrole
2.3.1.2 Polyaniline and Derivatives
2.3.2 Conductive Scaffolds Containing Inorganic and Metal Nanoparticles
2.3.2.1 Carbon Nanotubes
2.3.2.2 Graphene and Derivatives
2.3.2.3 Gold Nanoparticles
2.3.2.4 Silicon Nanowires
2.3.2.5 Mechanistic Exploration of Conductive Scaffolds Enhance Cardiac Tissue Regeneration
2.4 Conductive Scaffolds for Skeletal and Bone Regeneration
2.4.1 Carbon-Based Materials Used in Conductive Scaffolds
2.4.2 Conductive Polymers
2.5 Conductive Materials for Nerve Regeneration and Treatment
2.5.1 Conductive Polymers
2.5.2 Inorganic Nanoparticles
2.6 Wound Healing
2.7 Perspectives
References
Chapter 3: Nanotechnology in Dental Therapy and Oral Tissue Regeneration
3.1 Nanotechnology in Tooth Defect Therapy
3.1.1 Nano Tooth Filling Materials
3.1.2 Nano-Modified Adhesive Material
3.1.2.1 Enamel Bonding
3.1.2.2 Dentine Bonding
3.1.3 Nano Root Canal Filling Materials
3.1.4 Nano Enhanced Resin-Based Materials for Dentition Restoration
3.2 Nanotechnology in Oral Tissue Regeneration
3.2.1 Nanotechnology to Improve Stem Cells
3.2.2 Nanotechnology and Dental Stem Cells
3.2.3 Nanotechnology to Regenerate Keratinized Gingival
3.2.4 Nanotechnology to Regenerate Tooth
3.2.5 Shortcomings in the Current Application of Nanotechnology
3.3 Application of Antibacterial Nanomaterials in Dentistry
3.3.1 The Toxicological Side Effects in Nano-Incorporated Dental Materials and Device
References
Chapter 4: Next Generation of Cancer Immunotherapy: Targeting the Cancer-Immunity Cycle with Nanotechnology
4.1 Antitumor Immune Responses and Cancer Immunotherapy
4.2 Nanomaterials for Immunomodulation and Cancer Immunotherapy
4.2.1 Synthetic Nanomaterials
4.2.1.1 Organic Nanoparticles
4.2.1.2 Inorganic Nanoparticles
4.2.1.3 Organic-Inorganic Hybrid Nanoparticles
4.2.2 Biologically Derived Nanomaterials
4.2.2.1 Caged Protein-Based Nanoparticles
4.2.2.2 Cell Membrane-Based Nanoparticles
4.2.2.3 Immune Cell-Based Drug Delivery for Cancer Immunotherapy
4.3 Next Generation of Cancer Immunotherapy: Targeting the Cancer-Immunity Cycle with Nanotechnology
4.3.1 Enhancing Immunogenic Antigen Release with Nanomaterials (Step 1)
4.3.1.1 PDT-Triggered Immunogenic Antigen Release
4.3.1.2 PTT-Triggered Immunogenic Antigen Release
4.3.1.3 Chemotherapy-Triggered Immunogenic Antigen Release
4.3.1.4 Radiotherapy-Triggered Immunogenic Antigen Release
4.3.1.5 Oncolytic Virus-Triggered Immunogenic Antigen Release
4.3.2 Promoting Dendritic Cell-Targeted Immunotherapy with Nanomaterials (Step 2)
4.3.2.1 Nanomaterial-Based Cancer Vaccines
4.3.2.2 Facilitating APC-Targeted Vaccine Delivery with Nanomaterials
4.3.2.3 Promoting Antigen Cross-Presentation with Nanomaterials
4.3.2.4 Intrinsic Immunostimulatory Effect of Nanomaterials
4.3.2.5 Enhancing the Potency of Immunoadjuvants with Nanomaterials
4.3.3 Lymph Node-Targeted Cancer Immunotherapy with Nanomaterials (Step 3)
4.3.4 Reprogramming the Tumor Microenvironment with Nanomaterials (Steps 5 and 7)
4.3.4.1 Repolarizing Tumor-Associated Dendritic Cells with Nanomaterials
4.3.4.2 Targeting Tumor-Associated Macrophages with Nanomaterials
4.3.4.3 Targeting Myeloid-Derived Suppressor Cells with Nanomaterials
4.3.4.4 Targeting Regulatory T Cells with Nanomaterials
4.3.4.5 Targeting Immunosuppressive Factors with Nanomaterials
4.3.4.6 Ameliorating Hypoxia in Tumor Microenvironment with Nanotechnology
4.3.5 Targeting Antitumor Effector Cells with Nanomaterials (Step 6 and 7)
4.3.5.1 T Cell-Targeted Immunotherapy
4.3.5.2 NK Cell-Targeted Immunotherapy
4.3.6 Nanotechnology for Combinational Cancer Therapy
4.4 Perspectives
4.5 Conclusions
References
Chapter 5: Molecular Studies of Peptide Assemblies and Related Applications in Tumor Therapy and Diagnosis
5.1 Structures and Formation of Amyloid-Like Fibrils
5.1.1 Structures of Amyloid-Like Fibrils
5.1.2 Formation of Amyloid-Like Aggregates
5.2 Structural Perspectives of Peptide-Based Aggregates
5.2.1 Primary Peptide Structures with Aggregation Propensity
5.2.2 Nanostructures of Peptide Aggregates
5.3 Self-Assembled Peptide-Based Biomaterials
5.3.1 In Vitro Self-Assembled Peptide-Based Nanomaterials
5.3.2 In Vivo Peptide Assemblies for Tumor Diagnosis and Therapeutics
5.3.3 De Novo Design of Biologically Active Amyloid
5.4 Peptide-Based Drug Delivery Systems
5.4.1 De Novo Designed Peptides for Drug Delivery Systems
5.4.2 Peptide-Aggregate-Based Drug Delivery Systems
5.5 Conclusions
References
Chapter 6: Rationally Designed DNA Assemblies for Biomedical Application
6.1 Introduction
6.2 Self-Assembled DNA Nanostructures
6.3 DNA-Based Nanovehicles for Biomedical Application In Vitro and In Vivo
6.3.1 Chemotherapeutic Drugs
6.3.2 Functional Nucleic Acids
6.3.3 Nanoparticle
6.3.4 Proteins and Peptides
6.3.5 Dynamic DNA Nanodevices and Machines for Biomedical Application
6.4 Summary and Outlook
References
Chapter 7: Intermolecular Interactions and Self-Assembly of Peptide-Based Nanomaterials Against Human Pathogenic Bacteria
7.1 Introduction
7.2 Development of Peptide-Based Materials with Antibacterial Activity
7.2.1 Peptide Structures
7.2.1.1 Helices
7.2.1.2 β Structure
7.2.1.3 Other Nonrepetitive Structures
7.2.2 Self-Assembly of Peptides to Nanostructures
7.2.2.1 Tubular Nanostructures
7.2.2.2 Nanofibers
7.2.2.3 Spherical/Vesicle Structures
7.2.3 Structural Characterization of Self-Assembled Peptides
7.2.3.1 X-Ray Diffraction
7.2.3.2 NMR Spectroscopy
7.2.3.3 Cryogenic Electron Microscopy
7.2.3.4 Other Methods
7.2.4 The Application of Self-Assembled Peptide-Based Nanomaterials in the Diagnosis of Bacterial Infection
7.2.5 Application of Self-Assembled Peptide-Based Nanomaterials in the Treatment of Bacterial Infection
7.2.6 Encapsulation of Peptide by Nanomaterials Against Bacteria
7.3 Development of Peptide Mimetic Materials with Antibacterial Activity
7.3.1 β-Peptide: Molecular Structure and Conformational Stability
7.3.2 Intermolecular Interactions Encoded by β-Peptides
7.3.3 β-Peptide: Hierarchic Assemblies and Functional Properties
7.3.4 Nylon-3 Polymers Against Pathogens
7.4 Conclusions
References
Chapter 8: Nanomaterials and Reactive Oxygen Species (ROS)
8.1 ROS in Biology System
8.1.1 The Source of ROSs in Biology: Where the ROSs Produced?
8.1.2 The Regulation of ROS Production in Biology
8.2 Biological Effects Induced by Oxidative Stress from Nanoparticles
8.2.1 Antioxidant Defense
8.2.2 Inflammation
8.2.3 Cytotoxicity
8.3 Methods of Detecting ROS in Nanomaterials
8.3.1 ESR Technique
8.3.2 Optical Absorption
8.3.3 Spectroscopic Probe Technique
8.3.4 Nanoprobes for ROS Detection
8.4 Biomedical Applications of Nanomaterials by ROS Regulation
8.4.1 Cancer Therapy
8.4.2 Antibacteria
8.4.3 Prevention of ROS-Related Diseases
8.5 Outlook
References
Chapter 9: Protein Corona of Nanoparticles and Its Application in Drug Delivery
9.1 Introduction
9.2 Overview of Protein Corona
9.3 Characterization Techniques for Protein Corona
9.3.1 Isolation-Based Methodology
9.3.2 In Situ Measurements
9.3.3 Direct Imaging
9.3.4 Other Techniques
9.4 Influence of NP on Adsorbed Proteins
9.5 Influence of Protein Corona on NP
9.6 Influence of Protein Corona on Drug Delivery
9.6.1 Influence of Protein Corona on Drug Release
9.6.2 Influence of Protein Corona on Targeted Delivery
9.6.3 Influence of Protein Corona on Intracellular Delivery
9.6.4 Influence of Protein Corona on NP Toxicity
9.7 Exploring the Protein Corona for Drug Delivery
9.7.1 Exploiting Protein Corona to Load Drugs
9.7.2 Direct Modulation of Protein Corona to Improve Drug Delivery
9.7.3 Engineering NP to Tune Protein Corona for Drug Delivery
9.8 Challenges and Opportunities of Protein Corona in Drug Delivery
9.9 Conclusions
References
Chapter 10: Development of Realgar Nanotherapeutics for Cancer Treatments
10.1 Introduction
10.2 History of Use of Realgar in Medical Practices
10.2.1 Realgar in Traditional Chinese Medicine
10.2.2 Realgar in the Treatment of Cancer
10.3 Traditional Processing Strategy and Challenges
10.4 Development of Realgar Nanotherapeutics
10.4.1 Top-Down Approaches
10.4.1.1 Milling
10.4.1.2 Microfluidizer
10.4.2 Bottom-Up Approaches
10.4.2.1 Chemical Precipitation
10.4.2.2 Solvent Relay
10.4.2.3 Quantum Dots
10.5 Development of One-Step Preparation of Solid Dispersion
10.6 Therapeutic Effects of Realgar Nanoformulations in Cancer
10.6.1 Therapeutics Effects of Realgar Nanoformulations in AML
10.6.2 Therapeutics Effects of Realgar Nanoformulations in CML
10.6.3 Therapeutics Effects of Realgar Nanoformulations in Solid Tumor
10.6.4 Therapeutic Mechanisms of Realgar in Different Forms
10.7 Summary and Perspectives
References
Chapter 11: Fabrication and Applications of Magnetic Nanoparticles-Based Drug Delivery System: Challenges and Perspectives
11.1 Composition of MNPs Drug Carriers
11.2 Applications of MNPs
11.2.1 Drugs Carriers
11.2.2 Targeted Drugs Carriers
11.2.3 Magnetic Hyperthermia
11.2.4 MRI Contrast Agents
11.2.5 Tumor Theranostics
11.2.6 Nanozyme
11.2.7 Summary
11.3 Magnetic Drug Loading System
11.3.1 Magnetic Targeted Drug Carriers
11.3.1.1 Fabrication of Monodisperse IONPs
11.3.1.2 Fabrication of Elements-Doping IONPs
11.3.1.3 Fabrication of Core/Shell IONPs
11.3.2 Drug Loading Methods
11.3.2.1 Non-covalent Self-Assembly
11.3.2.2 Chemical Bonding
11.4 3S Effects of MNPs
11.4.1 Size Effects
11.4.2 Surface Effects
11.4.2.1 Small Molecular Compounds
11.4.2.2 Polymer
11.4.2.3 Natural Materials
11.4.3 Shape Effects
11.5 Perspectives and Future Challenges
References

Citation preview

Haiyan Xu Ning Gu  Editors

Nanotechnology in Regenerative Medicine and Drug Delivery Therapy

Nanotechnology in Regenerative Medicine and Drug Delivery Therapy

Haiyan Xu  •  Ning Gu Editors

Nanotechnology in Regenerative Medicine and Drug Delivery Therapy

Editors Haiyan Xu Department of Biomedical Engineering Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College Beijing, China

Ning Gu School of Biological Science and Medical Engineering Southeast University Nanjing, China

ISBN 978-981-15-5385-1    ISBN 978-981-15-5386-8 (eBook) https://doi.org/10.1007/978-981-15-5386-8 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Acknowledgments

The authors would like to thank for supports from the National Key Research and Development Program of China (2017YFA0205500, 2017YFA0205001), the National Natural Science Foundation of China (31571013, 21673055, 21773042, 31971295, 21790394, 31901007, 81471793, 81801771), the CAMS Innovation Fund for Medical Sciences (CIFMS 2016-I2M-3-004, CIFMS 2018-I2M-3-006, CIFMS 2019-I2M-1-004), the State Key Laboratory Special Fund (2060204), the Fundamental Research Funds for the Central Universities (3332019064, 2018PT31028), the Key Research Program of Frontier Sciences, Chinese Academy of Science (QYZDJ-SSW-SLH048), the Youth Innovation Promotion Association of Chinese Academy of Science (2018048), the Open Project Fund provided by Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS (NSKF201812), the Zhongyuan Thousand Talents Project (204200510016), the Program for Innovative Research Team (in Science and Technology) in the University of Henan Province (19IRTSTHN026), the Guangdong Natural Science Foundation of Research Team (2016A030312006), and the Shenzhen Science and Technology Program (JCYJ20160429191503002).

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Contents

1 Magnetically Actuated Scaffolds to Enhance Tissue Regeneration��������������������������������������������������������������������������������������������    1 Haiyan Xu, Suisui Hao, and Jiawei Zhou 2 Conductive Nanostructured Scaffolds for Guiding Tissue Regeneration��������������������������������������������������������������������������������������������   39 Haiyan Xu, Jie Meng, and Tao Wen 3 Nanotechnology in Dental Therapy and Oral Tissue Regeneration��������������������������������������������������������������������������������   91 Zukun Yang, Liping Han, Yu Guo, Lu Jia, Cheng Yin, and Yang Xia 4 Next Generation of Cancer Immunotherapy: Targeting the Cancer-­­Immunity Cycle with Nanotechnology������������������������������  191 Yifan Ma and Lintao Cai 5 Molecular Studies of Peptide Assemblies and Related Applications in Tumor Therapy and Diagnosis������������������������������������  255 Huayi Wang, Xiaocui Fang, Yanlian Yang, and Chen Wang 6 Rationally Designed DNA Assemblies for Biomedical Application������������������������������������������������������������������������������������������������  287 Qiao Jiang, Qing Liu, Zhaoran Wang, and Baoquan Ding 7 Intermolecular Interactions and Self-­Assembly of Peptide-Based Nanomaterials Against Human Pathogenic Bacteria ��������������������������  311 Wenbo Zhang, Lanlan Yu, and Chenxuan Wang 8 Nanomaterials and Reactive Oxygen Species (ROS)����������������������������  361 Tao Wen, Jianbo Liu, Weiwei He, and Aiyun Yang 9 Protein Corona of Nanoparticles and Its Application in Drug Delivery ��������������������������������������������������������������������������������������  389 Weiqi Zhang

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Contents

10 Development of Realgar Nanotherapeutics for Cancer Treatments������������������������������������������������������������������������������������������������  421 Tao Wang and Haiyan Xu 11 Fabrication and Applications of Magnetic Nanoparticles-Based Drug Delivery System: Challenges and Perspectives����������������������������  455 Fei Xiong and Yuxiang Sun

Abbreviation 1

124 polymer AFM AML APDCs APL ATRA BP BSA CA CD CEC CMC CNCs Col CR cryo-EM CS CTCs DM1 DTT EGCG EPR ESEM FDA Gel GelMA GGMA GS HA HA-SH

Poly(octamethylene maleate (anhydride) 1,2,4-butanetricarboxylate) Atomic force microscopy Acute myeloid leukemia Amphiphilic peptide-drug conjugates Acute promyelocytic leukemia All-trans retinoic acid Bispyrene Bovine serum albumin Cellulose acetate Circular dichroism N-carboxyethyl chitosan Carboxymethyl cellulose Cellulose nanocrystals Collagen Complete remission Cryo-electron microscope Chitosan Circulating tumor cells Maytansinoid Dithiothreitol (−)-epigallocatechin-3-gallate Enhanced permeability and retention Environmental scanning electron microscopy Food and drug administration Gelatin Methyl acrylic anhydride-gelatin Methacrylated gellan gum Gelatin sponge Hyaluronic acid Thiol-modified hyaluronic acid

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Abbreviation 1

x HCD HDAC hIAPP HLB HME HPLC IC50 IHC LbL MAAG MDS MEL MeTro MIDAS MNPs NFs NMR Nrp-1 OHA OPF P(VDF-­TrFE) PAA PAAm PAH PANi PCL PDA PDCs PDLLA PEDOT PEG PEGDA PEGS PG PGS PHEMA pI PLA PLGA PLL PLLA PNIPAAm PPY PSS PTAA

Hydrophobic central domain Histone deacetylase Human islet amyloid polypeptide Hydrophilic-lipophilic balance Hot melt extrusion High-performance liquid chromatography 50% inhibiting concentration Immunohistochemical Layer-by-layer Methacrylated aminated gelatin Myelodysplastic syndrome Melamine Methacryloyl-substituted recombinant human tropoelastin Metal ion-dependent adhesion site Magnetic nanoparticles Nanofibers Nuclear magnetic resonance Neuropilin-1 Oxidized hyaluronic acid Oligo(poly(ethylene glycol) fumarate) Poly(vinylidene fluoride-co-trifluoroethylene) Poly(acrylic acid) Polyacrylamide Poly(allylamine hydrochloride) Polyaniline Poly-ε-caprolactone or polycaprolactone Polydopamine Peptide-drug conjugates Poly(d, l-lactic acid) Poly(3,4-ethylenedioxythiophene) Poly(ethylene glycol) Polyethyleneglycol diacrylate Poly(ethylene glycol)-co-poly(glycerol sebacate) Polyglycolide Poly(glycerol sebacate) Poly(2-hydroxyethyl methacrylate) Isoelectric point Poly-dl-lactide or poly(lactic acid) Poly(lactic-co-glycolic acid) Poly-l-lysine Poly(l-lactide) Poly(N-isopropylacrylamide) Polypyrrole Poly(4-styrene sulfonate) Poly(thiophene-3-acetic acid)

Abbreviation 1 PU PVA PVDF PVP QCSG RIF ROS RTG SD SDS SF SPR SSM STM TCP TEAR ThT TKIs TNBC TPU TTR VEGFR2 VNTR κC

xi Polyurethane Polyvinyl alcohol Polyvinylidene fluoride Polyvinylpyrrolidone Glycidyl methacrylate functionalized quaternized chitosan gel Realgar-indigo naturalis formula Reactive oxygen species Reverse thermal gel Standard deviation Sodium dodecyl sulfate Silk fibroin Surface plasma resonance Sterically stabilized micelle Scanning tunneling microscopy Tissue-culture-treated polystyrene Transformation-enhanced accumulation and retention Thioflavin T Tyrosine kinase inhibitors Triple-negative breast cancer Thermal plastic polyurethane Transthyretin Vascular endothelial growth factor receptor 2 Variable number of tandem repeats κ-carrageenan

Abbreviation 2

Cell types Muscle cells

Myoblast cell Stem cell

Osteoblasts

Cell name Rat smooth muscle cells Cardiomyocyte/cardiac myocytes Neonatal rat ventricular myocytes Neonatal rat cardiomyocytes hiPSC-derived cardiomyocytes Embryonic-stem-cell-derived cardiomyocytes Mouse embryonic stem cell derived cardiomyocytes Primary cardiomyocytes Murine neonatal cardiomyocytes H9c2 rat cardiac myoblast cells C2C12 myoblast cells Human pluripotent stem cells Human pluripotent stem cells-derived cardiomyocytes Brown adipose-derived stem cells Human induced pluripotent stem cells Mouse embryoid bodies Mesenchymal stem cells Human embryonic stem cells Umbilical cord blood (UCB)-derived multipotent mesenchymal stem cells Stem cells Human mesenchymal stem cells Bone marrow derived stem cells Rat bone-marrow-derived mesenchymal stem cells Osteoblasts Osteosarcoma Mouse osteoblast cells

Abbreviation SMCs CMs NRVM / hiPSC-CMs / mES-CM / / H9c2 C2C12 hPS hPSC / hiPSCs Ebs MSCs hESC CB-hMSCs / hMSCs BMSCs MSCs / MG-63 /

xiii

Abbreviation 2

xiv Cell types Nerve-related cells

Stromal cell

Epithelial cell Blood-related cells

Cell name Nerve stem cells Rat pheochromocytoma 12 Neural stem cells Schwann cells Rat Schwann cell Dorsal root ganglion neurons Neuron Human neural stem/progenitor cells (hNSPCs) Central nervous system neurons Adipose tissue-derived stromal cells Fibroblasts Human cardiac fibroblast Mouse osteoblast cells Epithelial cells derived from breast adenocarcinoma Neutrophil Macrophage RAW 264.7 cells Platelet Endothelial cell

Abbreviation / PC12 / SCs RSC DRG neurons / / CNS neurons ADSCs / / / MCF-7 / / / / /

Chapter 1

Magnetically Actuated Scaffolds to Enhance Tissue Regeneration Haiyan Xu, Suisui Hao, and Jiawei Zhou

Abstract  Tissue defects beyond critical sizes and organ failure are great challenges in human health care. Biomaterial scaffold-guided tissue regeneration is one important and promising way to develop more effective therapeutic strategies to meet the clinical demands by creating microenvironments mimicking natural physiological conditions from the aspects of biochemistry and biophysics. The combination of nanotechnology in the biomaterial scaffolds have opened a new field in regenerative medicine and provided more powerful tools for developing strategies to modulate biochemical and mechanical microenvironments for tissue regeneration. In this chapter, we focused on the evolution and research progress of magnetic nanoparticles-­ based tissue engineering scaffolds. This chapter emphasized the synergistic effects of magnetic scaffolds in combination with external magnetic fields on cells differentiation and tissue regeneration through comprehensively reviewing the applications in multiple cells and tissues including bone, cartilage, cardiac, endothelial, nerve, Schwann cells, fibroblasts and macrophages, following the introduction of biological effects exerted by magnetic fields and magnetic scaffolds separately. The combination strategy allows for providing mechanical stimulations directly and remotely controls cells behaviors and fates in vivo without invasion, and provides promising platforms for drug screening in vitro and ex vivo. Keywords  Regenerative medicine · Tissue engineering · Magnetic scaffold · Mechanical stimulation · Remotely control

H. Xu (*) Department of Biomedical Engineering, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China e-mail: [email protected] S. Hao Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA J. Zhou Peking Union Medical College, Beijing, P. R. China © Springer Nature Singapore Pte Ltd. 2020 H. Xu, N. Gu (eds.), Nanotechnology in Regenerative Medicine and Drug Delivery Therapy, https://doi.org/10.1007/978-981-15-5386-8_1

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1.1  Introduction Magnetism is a long historical and intriguing physical cue that can alter the behaviors of a broad range of cells; therefore, the implementation of magnetic features in biomaterial scaffolds may offer innovative opportunities to control cell populations within engineered 3D microenvironments, with the potential to enhance their use in tissue regeneration or in cell-based therapies. For decades, magnetic nanoparticles, especially iron oxide nanoparticles, have attracted intense research interests in biomedical fields because the manufacture technologies are maturing, and they have shown promising perspectives in various applications, such as bio-separation, detection and diagnosis, medical imaging, thermal treatment against tumor, and targeting drug delivery [1–3]. In the past decade, a new application potential of magnetic nanoparticles in tissue engineering and regenerative medicine has been explored with increasing interests from the aspects of magnetically actuated mechanical stimulation and remote control, by integrating magnetic nanoparticles into substrates or implantable scaffolds to provide mechanical forces directly to cells grown on and inside. The application modality included magnetic scaffolds alone or in combination with external applied magnetic fields. Motivations of integrating magnetic nanoparticles into various matrices for uses in tissue engineering can be summarized into following aspects: First, magnetic nanoparticles would form magnetic phases in microscale that distribute in matrices. These aggregated nanoparticles would provide local magnetic moments or magnetic fields, which is assumed to be beneficial to the tissue regeneration according to the literatures of biological effects of magnetic fields. Second, the induction of magnetic nanoparticles brings magnetic property to scaffolds, which endows the scaffolds capacity of reloading with bioagents in situ after implantation. For example, magnetic scaffolds are expected to attract and take up growth factors, stem cells or other bioagents bound to magnetic nanoparticles in vivo by the guidance of an external magnetic field. The magnetic property of scaffolds can also be used to control microstructures of scaffolds (for example, to form aligned morphology) or to improve implant fixation and stability in  vivo, by the remote control of external magnetic fields. And third, implantable scaffolds containing superparamagnetic nanoparticles can be magnetized by responding to external applied magnetic fields, which allow for generating local strain-induced deformation to apply mechanical stimulations directly to cells grown on the scaffolds. This chapter will emphasize the synergistic effects of magnetic scaffolds in combination with external magnetic fields on cells differentiation and tissue regeneration, through comprehensive reviewing the applications in multiple cells and tissues including bone, cartilage, cardiac, endothelial, nerve, Schwann cells, fibroblasts, and macrophages, following the introduction of biological effects exerted by magnetic fields and magnetic scaffolds separately. The combination strategy allows for providing mechanical stimulations directly and remotely controls cells behaviors and fates in vivo without invasion and provides promising platforms for drug screening in vitro and ex vivo.

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1.2  B  iological Effects of Magnetic Fields on Tissue Regeneration Magnetic fields include static (SMF) and electromagnetic fields (EMF). The therapeutic functions of magnetic fields have attracted clinical and research attentions for many years [4]. The SMF is usually generated by permanent magnetic materials, and there are various methods for obtaining the electromagnetic field generated by an arbitrarily moving point charge in free space. The EMF can be subdivided into pulse electromagnetic fields (PEMFs), alternating magnetic field (AMFs), or rotating magnetic fields (RMFs). Among them, SMFs and PEMFs are used most widely. According to the field intensity, SMF can be classified into weak field (less than 1 mT), moderate field (1 mT–1 T), strong field (1–5 T), and ultra-strong field (>5 T). It has been reported that the magnetic field with moderate intensity could enhance pre-osteoblasts proliferation and ECM production, and templated mineral deposition more rapidly than the control culture [5]. As an example, exposure to SMF of 160–340 mT enhanced the bone formation of rat calvaria cells [6] through the activation of p38 phosphorylation at the cellular level to stimulate osteoblastic differentiation [7]. There are also literature reports about the effects of magnetic fields on nerve system. For example, low-frequency magnetic fields could activate N-methyl-­ d-aspartate receptors in glutamatergic Ca2+ channels to promote neuronal differentiation [8] and might improve depressant behavior and cognitive dysfunction mainly by modulating synaptic function [9]. Impacts of the magnetic stimulation on human mesenchymal stem cells (hMSCs) have also been explored, as hMSCs are widely accepted as seeding cells, and the control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. A group reported that equine adipose-derived mesenchymal stem cells (ASCs) exhibited higher mRNA levels of bone morphogenetic protein­2/4 (BMP-2/4), Sox9, and type I collagen when cultured under the magnetic field of 0.2 T. In addition, the cells cultured in the magnetic field reached the population doubling time earlier, because the colony-forming potential of equine ASCs was higher when cells were cultured under magnetic field conditions. Interestingly, exposure to the magnetic field increased the number of secreted microvesicles in comparison to the control culture as well as increased the production of growth factors [10]. Another study reported the effect of low-frequency EMF on hMSCs during chondrogenic differentiation, the cultures were exposed to the low-frequency magnetic fields (5 mT) [11]. It was observed that the effect of EMF on collagen type II expression was cell passage-dependent and exclusively detected at higher cell passages. Under the EMF and the addition of human fibroblast growth factor 2 (FGF-2) and human transforming growth factor-β3 (TGF-β3), hMSCs showed a significant increase in collagen type II expression at passage 6, indicating that the very low-frequency EMF was able to improve the growth factor-induced chondrogenic differentiation of hMSCs, however, when growth factors were absent, there was no staining detected for safranin-O and collagen type II, indicating that the EMF could not induce chondrogenic differentiation alone. SMF exposure could

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also affect the cell viability and capacity of osteogenic differentiation in rat dental pulp cells (DPCs) [12]. It is interesting to note that exposure to SMF alone did not affect the dental pulp cell proliferation, only when exposed to SMF with the supplementary Dex/b-GP, the cell differentiation was significantly enhanced at the seventh and ninth day of culture when compared with the control culture, which was speculated that the magnetic field played a cooperative role via the ECM-mediated activation of ERK1/2-Cbfa1 signaling, while the new ECM deposition was time consuming. Another study showed that the magnetic field could stimulate biological functions of MSC; however, the responses of cells depended strongly on the substrate to which they adhere and on the crosstalk between integrin-mediated signals and soluble factors [13]. The results again implied that the magnetic stimulation alone on cells is not enough for the bone repair and regeneration, the microenvironment where the bone cells live is likely to play crucial synergy roles in the regeneration process. Another group had MSCs seeded in a composite scaffold composed of hydroxyapatite and collagen I, the construct was placed in a dynamic perfusion bioreactor and exposed to EMF of 15 Hz/1 mT. The applied EMF could promote osteogenic differentiation with the inducing medium. When implanted in a defect hole of 4 mm in width and 8 mm in depth that was created in each lateral femur condyle of rabbits, micro-CT results revealed that EMF could promote integration at bone–implant interface and bone regeneration after 12 weeks [14]. These results implied that scaffolds might be necessary as media for magnetic fields to elicit promoting effects on MSCs differentiation.

1.3  B  iological Effects of Magnetic Materials on Tissue Regeneration Inspired by the biological effects elicited by the low or moderate intensity of magnetic fields, it has been considered that magnetic materials may have positive effects on scaffold-guided tissue regeneration as well, because magnetic materials usually consist of iron (Fe), cobalt (Co), or nickel (Ni), and these materials can produce magnetic fields directly or indirectly, as it is well known that permanent magnets produce static magnetic fields (SMFs). As examples of the efforts, the roles of magnetic scaffolds in the guidance of bone tissue regeneration have been investigated largely. In case of degenerative disease or lesion, bone tissue replacement and regeneration are an important clinical goal. In long period the repair for critical size defects rely on implantable grafts that are not biodegradable. The emerging of tissue engineering and regenerative medicine provide the concept of developing biodegradable scaffolds that are 3D structural supports, allowing cellular infiltration and subsequent integration with the native tissue. Several ceramic hydroxyapatite (HA) scaffolds with high porosity and good osteointegration have been developed in the past few decades but they have not solved completely the problems related to bone defects, a major challenge is the speed of scaffold-induced new bone formation is

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too low to meet the clinical demand [15, 16]. Researchers have been making great efforts to overcome the problem; the utilization of magnetic nanoparticles is one of the directions. Early investigations mainly addressed the biocompatibility of magnetic nanoparticles incorporated in matrices. For example, researchers developed a simple and inexpensive technique able to transform standard commercial scaffolds made of hydroxyapatite and collagen into magnetic scaffolds by dip-coating the scaffolds in aqueous ferrofluids containing iron oxide nanoparticles coated with various biopolymers. By this way, the nanoparticles were integrated into the structure of the scaffolds, providing the latter with magnetization values as high as 15  emu/g at 10 kOe [17]. These values were considered suitable for generating magnetic gradients, enabling magnetic guidance in the vicinity and inside the scaffold. It was reported that the magnetic scaffolds did not suffer from any structural damage during the process, maintaining their specific porosity and shape. Moreover, they do not release magnetic particles under a constant flow of simulated body fluids over a period of 8 days, indicating the good stability of the scaffold. Computer simulations revealed that the magnetic field generated by magnetic scaffolds was ready within the useful range for in vivo applications. Cellular experimental results showed that the composite scaffold supported the proliferation of human bone marrow-derived stem cells (HBMSCs), the cells covered the whole surface and filled most of the macropores of scaffolds, and no statistically significant differences in cellular viability and osteoblastic differentiation among all types of scaffolds at each time point were observed. Furthermore, the composite of hydroxyapatite (HA) dipping magnetic fluids was tested in a magnetic field of 320 mT, no negative effects arising from the presence of magnetite [18]. A comprehensive comparison study for the bone tissue formation was conducted by the same group in a preclinical rabbit model of critical femoral defect treated either with a HA/magnetite (90/10 wt%) or pure HA porous scaffolds at 4 and 12 weeks after implantation, indicating that both scaffolds were able to allow bone regeneration, and that the HA scaffold dipping magnetic fluids did not have short-term adverse effects on biocompatibility and bone formation ability [19]. Another pioneer work is to fabricate magnetic composites that were composed of CaP ceramic and magnetic nanoparticles (CaP-MNP). In the fabrication, superparamagnetic nanoparticles were integrated to two kinds of CaP ceramics, one was hydroxyapatite (HA), the other one was a composite of HA and tricalcium phosphate in a ratio of 65:35, named HT. When bone-related cell line Ros17/2.8 and MG63 cells were seeded in the composites and incubated for 4 or 7 days, the cell proliferation was significantly promoted compared with that of nonmagnetic scaffolds. The possible influence of magnetic materials on bone morphological protein (BMP) was concerned in this investigation. BMP-2 was loaded on the magnetic scaffolds that were implanted subcutaneously in the fasciae of rat back muscles for 30 days. The in  vivo test showed that the expression of BMP-2 was accelerated by HT containing superparamagnetic nanoparticles, and new bone-like tissue formation was observed, indicating the function of loaded BMP-2 was not affected by the incorporation of superparamagnetic nanoparticles. Importantly, it was observed that ALP secretion of Ros17/2.8 cells was increased on the magnetic

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composites more than those on nonmagnetic composites after 14 days incubation, but the magnetic composites did not affect the ALP secretion of MG63 cells. Those results showed an important cue that the CaP-MNP composites could promote osteogenetic differentiation as well as the proliferation [20]. In addition to inorganic scaffolds, iron oxide nanoparticles are widely employed in polymer-based scaffolds, aiming to improve the biocompatibility and mechanical strength. One typical example is that a nanofibrous scaffold made by electrospinning technique from a mixture of poly-ε-caprolactone (PCL) and γ-Fe2O3 nanoparticles of 23 nm in diameter. The scaffolds obtained the magnetic responsive property, which contributed to the enhancement of adhesion and proliferation of porcine mesenchymal stem cells. Importantly, the PCL-based scaffold-induced osteogenic differentiation of mesenchymal stem cells, which was evidenced by the increased ALP activity [21]. Similarly, magnetic nanofibrous scaffolds of PCL incorporating citrated magnetic nanoparticles (MNP) of 12  nm in diameter were fabricated by using electrospinning technique, the content of MNPs was up to 20% in the solution. The incorporation of MNPs greatly improved the hydrophilicity of the nanofibers as well as enhanced the tensile mechanical properties of the nanofibers. In particular, the tensile strength increase was as high as 25 MPa at 15% MNPs, much higher than 10 MPa for pure PCL. The PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation rate of the PCL nanofibers in a content dependent manner. When subcutaneously implanted in rats, the composited nanofibers significantly improved initial cell adhesion and subsequent penetration through the nanofibers for rat bone marrow MSCs, compared to the pure PCL control, and increased the alkaline phosphatase activity and the gene expression of collagen I, osteopontin, and bone sialoprotein. Noted that they induced substantial neoblood vessel formation which greatly limited in the pure PCL, while exhibited minimal adverse tissue reactions [22]. The same formulation of PCL and MNPs was processed into porous scaffolds by using salt-leaching approach, which also showed the superparamagnetic behavior. When applied on bone repair, similar results were obtained, confirming the effects observed in the nanofibrous scaffolds. As shown that the incorporation of MNPs greatly improved the hydrophilicity and water swelling of scaffolds. More mineral nanocrystallites covered on the surfaces of the composite scaffolds than on the PCL scaffolds in the in vitro acellular mineralization experiment. The mechanical stiffness increased significantly with the addition of MNPs when tested under both static and dynamic compressed wet conditions. The subcutaneous implantation in rats for 2 weeks revealed favorable tissue compatibility, with substantial fibroblastic cell invasion and neoblood vessel formation while exerting minimal inflammatory reactions in the testing groups [23]. From these investigations we can notice the important roles of iron oxide nanoparticles in the scaffolds for enhancing bone tissue regeneration. When make further comparison with data from above references, it seemed the smaller the nanoparticles (23 nm vs. 12 nm), the stronger the enhancing effects. Another example is magnetic PLGA/PCL scaffolds that were fabricated using a combination of the electrospinning technique and layer-by-layer

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assembly of superparamagnetic iron oxide nanoparticles (IONPs) greatly enhanced the hydrophilicity and increased the elastic modulus of the scaffolds. At the early stage of cell culture, the adhesion and spreading of ADSCs of IONPs group were more stretched and spindle-like compared to those on the control groups, which indicated that IONPs-containing scaffolds affected cellular behavior at an early stage. It could be noticed that IONPs group induced ADSCs osteogenic differentiation, evidenced by the highest ALP activity. Higher mineralization rate also confirmed the importance of magnetic nanomaterials as a bioactive interface between cells and scaffolds [24]. Chitosan and collagen are two kinds of biological materials widely investigated for scaffolds fabrication. A group prepared composite scaffolds containing hydroxyapatite nanoparticles (nHAP) and Fe3O4 nanoparticles (Fe3O4 NPs), and the substrate of the scaffolds was the mixture of chitosan and collagen (CS/Col). It is interesting that during the cell culture, a mass of hydroxyapatite microparticles could be observed forming on the surfaces of scaffolds containing Fe3O4, whereas did not on the surface of CS/Col/nHAP, suggesting that Fe3O4 NPs enhanced mineralization. The composite scaffolds (CS/Col/Fe3O4/nHAP) not only showed superior structural and mechanical performance for pre-osteoblasts cell line MC3T3-E1 cells adhesion and proliferation, but also had a higher bone regeneration ability when implanted into the SD rats’ skull defects comparing to control group [25]. In another kind magnetic scaffold composed of HA, collagen, and magnetic nanoparticles, the magnetic phase could act as a sort of cross-linking agent for the collagen, inducing a chemical-physical-mechanical stabilization of the material and allowing for the control of the porosity network of the scaffold, while the hydroxyapatite (HA) was directly nucleated onto collagen fibers during their self-assembly, representing the biomineral calcium phosphate phase [26]. Gelatin is also one widely applied biological material in tissue engineering. When integrated with superparamagnetic nanoparticles (SPIONs), the magnetic gelatin sponge (GS) was implanted in the incisor sockets of Sprague-Dawley rats sacrificed at 2 and 4 weeks. In addition to the enhancement of the bone regeneration, the scaffold degradation and interaction with host tissues could be visually monitored over time by MRI, and a significant decrease in the signal intensity of T2-weighted magnetic resonance imaging (MRI) was also observed. This is another advantage of superparamagnetic iron oxide nanoparticles, the residual SPIONs, together with newly formed bone [27]. Magnetic composites also showed application potentials in the treatment of tumor related-bone defects. For example, magnetic SrFe12O19 nanoparticles modified-­mesoporous bio-glass (BG)/chitosan (CS) porous scaffold (MBCS) not only triggered tumor apoptosis and ablation by elevating the temperatures of tumors under the irradiation of near-infrared (NIR) laser, because the SrFe12O19 nanoparticles in MBCS improved the photothermal conversion property, but also promoted the expression levels of osteogenic-related genes (OCN, COL1, Runx2, and ALP) and the new bone regeneration by activated BMP-2/Smad/Runx2 pathway, indicating the osteogenic differentiation was effectively induced by the magnetic composite nanoparticles. In this process, the residual SPIONs and newly formed bone could be monitored in the treatment of tumor related-bone defects [28]. Another example

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is the fabrication of 3D porous Mg2SiO4-CoFe2O4 nanocomposite scaffolds with interconnected porosity and desirable mechanical properties close to cancellous bone, loading chemotherapeutics for bone cancer treatment. When exposed with electromagnetic fields of 100, 125, 150, and 200 Oe under 200 kHz, the scaffolds could produce heat for hyperthermia-based chemotherapies and displayed good biocompatibility with MG63 cells [29]. Nevertheless, it should be noted that there were literatures reporting no osteogenestic differentiation was induced by polymeric magnetic scaffolds. For instances, the induction of osteogenestic differentiation was not detected upon the electrospun membrane of integrating magnetic nanoparticles of Fe3O4 into biodegradable chitosan (CS) and poly vinyl alcohol (PVA), though human osteoblast-like cells MG63 grew well on the composite scaffold [30]. The electrospun membrane composed of poly(lactic-co-glycolic acid) (PLGA) and hydrophobic superparamagnetic nanoparticles (MNPs) showed good biocompatibility and significantly promoted cell proliferation compared with PLGA nanofibrous scaffold. Again, the composite scaffolds had no effects on the differentiation of MC3T3-E1 cells [31]. As for the dental tissue regeneration, magnetic scaffolds are being investigated in recent years, which is still an unmet clinical need. So far, no therapies have been completely successful in regenerating dental tissue complexes such as periodontium. One of the major challenges is the lack of scaffolds that can guide and direct cell fate toward the reconstruction of different mineralized and nonmineralized dental tissues. The osteogentic effect was reported by using magnetic scaffolds in some investigations. For example, composite foam-like scaffolds of PCL and Fe3O4 nanoparticles were fabricated by a salting out procedure, there were pores ranging from 250 to 500 μm inside of the scaffold, the size distribution was considered suitable for cellular population and odontogenic differentiation. When human dental pulp cells (HDPCs) were cultured on the magnetic composite scaffolds containing Fe3O4 nanoparticles up to 10 wt%, they exhibit good viability. It is interesting that the migration rate of the cells was enhanced after incubated with the composite scaffolds, determined by the in vitro scratch assay. At the same time, the adhesion of HDPCs was significantly promoted. Furthermore, the magnetic scaffolds significantly influenced the odontogenic differentiation of HDPCs, supported by the increased ALP activity, mRNA expression of odontogenic markers, and mineralized nodule formation. Mechanisms may include the upregulation of integrin subunits of α1, α2, β1 and β3, and the activation of downstream pathways including FAK, paxillin, p38, ERK MAPK, and NF-κB [32]. The magnetic scaffolds with nanofibrous structures were also showed similar positive effects on the human dental pulp cell (HDPCs) behaviors. The growth of HDPCs was significantly enhanced on the magnetic scaffolds compared with that on the nonmagnetic counterpart. The odontogenic differentiation of cells was significantly upregulated by the culture with magnetic scaffolds. Furthermore, the magnetic scaffolds promoted the HDPCs-­ induced angiogenesis of endothelial cells. Mechanistic studies indicated that the expression of signaling molecules, Wnt3a, phosphorylated GSK-3β and nuclear β-catenin, was substantially stimulated by the magnetic scaffolds; in parallel, the MAPK and NF-κB were highly activated when cultured on the magnetic nanofiber scaffolds [33]. Considering that tooth is a complex organ made of highly

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mineralized tissues where the nonmineralized periodontal ligament (PDL) connects the alveolar bone to the cementum and ensures tooth functionality and stability, a very compact scaffold was made by integrating three layers, one was the bone-like layer, consisting iron-doped hydroxyapatite (FeHA), acting as mineral phase nucleated on collagen fibers; the second one was a ligament-like layer consisting of collagen, the third one was the cementum-like layer consisting of FeHA and cellulose acetate (CA), preserving its dense morphology. The scaffold was endowed with a superparamagnetic ability and showed that the chemical composition and morphology of the scaffolds favor substantial cell viability and proliferation [34]. Magnetic nanoparticles are also utilized as carriers of loading growth factors in the treatment of spinal cord injuries and might act together as imaging contrast to monitor the healing process by MRI. For example, free bFGF has short half-life of about 3–10 min due to rapid enzymatic degradation, this disadvantage leads to loss of biological activity and functions. A magnetic fibrin hydrogel scaffolds were prepared by blending thrombin-conjugated γ-Fe2O3 nanoparticles with fibrinogen to form magnetic fibrin hydrogel, with the addition of magnetic nanoparticles conjugated with basal fibroblast growth factor (bFGF). The composite hydrogel was applied to human nasal olfactory mucosa (NOM) cells that are primary cells of interest for implantation into spinal cord injuries. The conjugated bFGF enhanced the growth and differentiation of the NOM cells in the fibrin scaffolds significantly, compared to the same or even five times higher concentration of the free bFGF. In the presence of the bFGF-conjugated magnetic nanoparticles, the cultured NOM cells proliferated and formed a three-dimensional interconnected network composed mainly of tapered bipolar cells [35]. Data collected from some of above investigations were summarized in Table 1.1. It is seen that all the magnetic scaffolds showed biocompatibility, in some cases magnetic scaffolds alone did not induce osteogenesis though some did. It seems whether magnetic scaffolds could induce osteogenic differentiation or maturation is largely involved with the cell type and the chemistry and microstructures of scaffolds. And should be noted that even the osteogenesis was induced by magnetic scaffolds, there was still a large space for the degree to increase.

1.4  C  ombination of Magnetic Scaffolds and Magnetic Fields to Accelerate Tissue Regeneration The combination of magnetic scaffolds and magnetic fields has emerged since 2010. This strategy’s aim is to provide stronger and remotely controllable stimulations to bone-related cells to enhance osteogenesis and new bone formation. In recent years, this combination strategy has gained increasing research interests and has been demonstrated effective to multiple cell type including bone, cartilage, endothelial, nerve, fibroblasts, macrophages, and Schwan cells. Data from some investigations are summarized in Table  1.2, the magnetic component, magnetic field strength, scaffolds composition, and biological effects were emphasized.

Electrospinning

Fe3O4 NPs (12 nm)

PCL

Casting

Rapid Prototyping

PCL/FeHA

Iron-doped hydroxyapatite (FeHA)

Magnetic nanoparticle Hydroxyapatite (Mgn)

Salt-­leaching

PCL

Fe3O4 NPs (10.7 nm)

in situ crystallization and freeze-drying

Hydroxyapatite nanoparticles, Chitosan and Collagen

Other components Preparation methods Hydroxyapatite and/ Foamed and sintered, or Ca3(PO4)2 followed by immersed into Fe3O4 NPs colloid

Fe3O4 NPs

Magnetic component Fe3O4 NPs (5 nm)

Table 1.1  Magnetic scaffolds and biological effects on cells

Nanofibrous

Porous

3D porous

Porous

Porous

Structure Porous

Biological effects Enhanced proliferation and ALP activity increased; new bone-like tissue formation observed Enhanced adhesion, proliferation, and differentiation; better tissue compatibility and higher bone regeneration ability MC3T3-E1; subcutaneous Enhanced proliferation, implantation of SD rats differentiation, and mineralization Favorable tissue compatibility, neoblood vessel formation, minimal inflammatory reactions Bone marrow mesenchymal Promote adhesion, proliferation, and stem cells (hBMSCs) Implantation in critical bone differentiation; new bone formation after 4 weeks defect in lateral femoral condyle of male rabbits Saos-2; critical bone defect Enhanced proliferation in lateral femoral condyle of Good compatibility and bone regeneration in vivo male rabbits Promote adhesion, Rat bone marrow penetration and mesenchymal stem cells differentiation (MSCs) Small subcutaneous pouches Induce neoblood vessel formation and bone in the back area laterally regeneration from the spine of SD rats

Cell and animal model Ros17/2.8; MG63; implanted in the skin incision at the midline of the SD rat back MC3T3-E1; Full-­thickness defects with diameter of 5 mm on the middle ridge of skull in SD rats

[7]

[5]

[10]

[8]

[17]

Refs. [1]

10 H. Xu et al.

PCL

Commercial magnetic NPs (50–100 nm)

3D porous 3D macroporous 3D sphere

Sol-gel Electrospinning and layer-by-layer assembly Covalent conjugation and co-precipitation One-pot hydrothermal and post-reduction annealing

PLGA/PCL

Chitosan

Calcium phosphate

Fe3O4 NPs

FeCaP ( 10,000 Oe) and a coercive field Hc of 0.481 kA/m. MNPs-free-­ κChydrogels were prepared and considered as reference materials. In this study, a magnetic stimulation of 350  mT applied to human adipose-derived stem cells (hASCs) grown on the magnetic kC hydrogels through an oscillation frequency of 2 Hz. It was shown that the concentration of MNPs in the hydrogels influenced cellular behavior, tuning a positive effect on cell viability and metabolic activity of hASCs, and the most promising outcomes were observed in 5% MNP-κC matrices. Although hASCs laden in MNPs-free- and MNPs-κC hydrogels showed similar metabolic and proliferation levels, the mRNA transcripts of Collagen I, Collagen II, and Sox9 was upregulated more, pronounced by Day 14  in hASCs laden in MNPs-κC hydrogels under the magnetic field, suggesting that the magnetic stimulation induced an earlier and stronger upregulation of chondrogenic-specific transcripts. In particular, the enhanced expression of Sox9 suggests that the combination of MNP-κC hydrogel and magnetic stimulation plays a role in the regulation of the chondrogenic commitment of hASCs, especially in chondrogenic cultures [57]. In order to develop scaffolds mimicking the depth-dependent material properties of native articular cartilage, a research group fabricated a tri-layered magnetic composite agarose gel (ferrogel) with a gradient in compressive modulus from the top to the bottom layers of the construct. The three layers have different weight percentages of dextran-coated iron oxide nanoparticles 20%, 7%, and 10%. Rare earth NdFeB magnets were used to generate an external magnetic field (0.4, 0.5, or 0.75  T). The ferrogel was able to respond to the external magnetic stimulation, exhibiting an elastic, depth-dependent strain gradient. When bovine chondrocytes (BCs) were seeded in the ferrogel scaffolds and cultured for up to 14 days, it was shown that sulfated glycosaminoglycan increased with the depth from the surface and was more significant at the 12 days of culture, indicating a biochemical gradient was formed, and meanwhile good cell viability was detected [58]. In another study, agarose hydrogels containing Fe3O4 nanoparticles were seeded with human mesenchymal stem cells and placed under external magnetic fields. By this way the

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hydrogels provided a pressureless, soft mechanical stimulation. The study presented that the magnetic hydrogels under magnetic fields could induce significant chondrogenesis of mesenchymal stem cells without any additional chondrogenesis transcription factors, with the upregulation of aggrecan, SOX9, and collagen II in protein level, and the effect could be remarkably enhanced by the supplementary of TGF-β1 or dexamethasone. In addition, the movement frequency of the magnetic hydrogels was investigated as well to figure out the optimal parameters [59]. In another example, to closely mimic the hierarchical architecture and biomechanical behavior of native tendons, continuous and aligned electrospun fiber threads were fabricated using PCL matrix filled with hybrid rod-shaped cellulose nanocrystals (CNCs) decorated with iron oxide MNPs (MNP@CNC). Human adipose stem cells (hASCs) were grown on the magnetic scaffolds under magnetic stimulating conditions (oscillation frequency of 2 Hz and 0.2 mm of horizontal displacement) for 21 days, which displayed higher degrees of cell cytoskeleton anisotropic organization and steered the mechanosensitive YAP/TAZ signaling pathway. As consequent results, the stimulated cells showed increased expression of tendon-related markers and pro-healing cytokines and lowered inflammatory cytokines in gene level [60]. An interesting and a little complex design is 3D porous magnetic microbead. The main body was microspheres of poly(lactic-co-glycolic acid) (PLGA) with gelatin beads distributing inside. When the gelatin beads were removed, the microsphere became porous while magnetic nanoparticles were conjugated to the surface of the walls via amino bond formation. This composite microscaffold was used to the culture of D1 mouse MSCs to increase adhesion, migration and chondrogenic differentiation under magnetic fields, and could be magnetically guided to the target area by the magnetic field guidance, for example, to the medial condyle of the femur in the knee joint model created by a 3D printer [61].

1.4.3  Endothelial, Cardiac, and Neuron Cells Vascularization is one of the major challenges in engineering tissues, especially thick and complex tissue such as cardiac muscle, which would enable the engineering tissue efficiently integrated with host tissue upon implantation. For example, the vascularization is one of the greatest hurdles hindering the successful implementation of tissue-engineered cardiac patch as a therapeutic strategy for myocardial repair. A group investigated the effect of magnetite-impregnated alginate scaffolds on aortic endothelial cells. The magnetic field was applied during the first 7 days to stimulate the cells out of a total 14-day experimental course, which was set up by passing an electrical current through serially connected coils at frequency of 40 Hz. The scaffold was generated by a freeze-dry technique from a mixture of alginate stabilized ferro-fluid and alginate to obtain 1.2 (w/v)% of final concentration of magnetite post-crosslinking while the control was fabricated by the same method without magnetite. The investigation showed that the employed strategy significantly elevated the metabolic activity of the endothelial cells during the stimulation

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period and suggested that the elevated metabolic activity was related to the cell migration and re-organization. Although the magnetic stimulation alone has the positive effect on the activity of cells grown on the nonmagnetic scaffold, the magnetic hydrogel significantly further enhanced this effect. The study also observed that cellular vessel-like (loop) structures were formed on day 14 in the magnetically stimulated scaffolds without supplementation of any growth factors, while little was observed in the non-stimulated (control) scaffolds. The loop structure is known as indicators of vasculogenesis and angiogenesis, therefore this study provided strong evidence that the magnetically actuated scaffolds could enhance the vascularization of engineered tissues, which indicated possibility of fabricating pre-­vascularized tissue construct with potential applicability for transplantation [62, 63]. Magnetically actuated scaffolds were also used to generate strain stimulus on rat aortic smooth muscle cells. Interestingly the strategy of inducing magnetic scaffolds to generate deformation under the magnetic field was utilized to improve cell viability in the scaffold interior by pumping nutrients throughout the structure. The magnetic scaffolds were fabricated in a tube shape by winding electrospun sheets of a biodegradable polymer 50/50 blend of polyglycolide (PG) and polycaprolactone (PCL), in which were dispersed magnetic γ-Fe2O3 nanoparticles. The tubular scaffolds were seeded with smooth muscle cells and actuated by a static magnetic field to induce a cyclic crimping deformation, permanent magnets mounted on actuators were used to apply a cyclically varying magnetic field to each culture and magnetic actuation was cycled for 60 s on and 60 s off to ensure adequate time for deflection and recovery for a time period of 1–15 days, which applied strain stimulus to the cells and pumped nutrient fluid through the porous tube walls. Results were that the magnetically actuated scaffolds significantly increased the secretion of Type I collagen that is one of the most abundant ECM proteins and synthesized by vascular SMC. On the contrary, collagen production of non-actuated scaffolds was minimal at the same time point compared with the actuated scaffolds [64]. A group built a functional cardiac patch by combining the use of a macroporous alginate scaffold impregnated with magnetically responsive nanoparticles (MNPs) and the external magnetic stimulation. Researchers seeded neonatal rat cardiac cells on the magnetically responsive scaffolds that were stimulated by an alternating magnetic field of 5 Hz. It was shown that a short-term stimulation of 20 min external magnetic field increased the phosphorylation of AKT in the cardiac cell constructs, meanwhile there was a greater extent of striated fibers in the stimulated group than in the non-­ stimulated group, and the level of contractile protein Troponin-T and adhesive conjunction protein Connexin-43 was higher in the stimulated group than that in the control group on day 15. These results indicated that a synergistic effect of magnetic field stimulation together with the magnetic responsive scaffolds provided a favor regenerating environment for cardiac cells, driving their organization into functionally mature tissue [65]. Nerve regeneration and recovery of nerve function have been a major issue in neuroscience. For the regeneration of peripheral nerves, functional recovery is totally dependent on the growth and extension of axons from the proximal end across the injured site until they reach their distal target. Because rate of axonal

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regeneration in humans occurs about 2–5 mm/day, it can take many months to get recovery of the functions for significant complete injuries, such as neurotmesis. Great efforts have been made to develop more effective approaches for accelerating the recovery time. One approach is the “guidance therapy” based on the use of scaffolds working as nerve guidance conduit for the diffusion of growth factors secreted by the injured nerve ends and to limit the injury site infiltration by scar tissue. Recently the importance of physical stimuli for neuronal growth and development has been widely recognized and appealing in literatures. Specifically, results from published experimental studies indicate that carefully controlled forces can modulate neuronal regeneration. Based on above knowledge, roles of magnetic scaffolds have been explored in remotely controlling nanofibers orientation in scaffolds is important for neuronal cells growth. A research group proposed a noninvasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields. In the study, researchers synthesized MNPs functionalized with nerve growth factor beta (NGF-β), which were applied to neuron-like cell line PC12 cells in combination with a magnetic field to validate the concept that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction. The magnetic nanoparticles were used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. They have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction, as well as demonstrated that this approach could trigger PC12 differentiation in a neuronal phenotype [66]. A group reported another interesting design. Magnetic electrospun nanofibrous membranes were generated with the solution of PLLA and magnetic nanoparticles, which were cut into small pieces and rolled into conduits of 10 mm in length. The shortened conduits were next injected with collagen hydrogel in situ. Here the magnetic responsive property of scaffolds was utilized to reposition the small conduits dispersing in the hydrogel. A magnetic field was applied to guide the conduits aligned in situ. This injectable guidance system provided consistent topographical guidance to cells, increased both neurite alignment and neurite length within the hydrogel scaffold, neurites that contacted the fiber conduits followed the orientation of the fibers [67]. By integrating magnetic nanoparticles into collagen hydrogels, the gel orientation could be controlled dynamically and remotely in situ under an external magnetic field. In this design, magnetic particles aggregated into magnetic particle strings during the gelation period, leading to the alignment of the collagen fibers. Results showed that neurons within the 3D gels exhibited normal electrical activity and viability. Importantly, neurons formed elongated and co-oriented morphology under the guidance of the particle strings and fibers as supportive cues for growth [68]. A similar design was given below. To overcome the lack of the required structural complexity to regenerate aligned tissues, an injectable rod-shaped, magnetoceptive microgel was prepared by subjecting the prepolymer solution within a magnetic field before molding and curing and interlocking the oriented microgels. The obtained hybrid hydrogel with global unidirectional anisotropy was applied to primary dorsal root ganglions (DRGs), showing a strong guidance effect [69].

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1.4.4  Immune Cells and Supportive Cells Macrophages play crucial roles in various immune-related responses, such as host defense, wound healing, and tissue regeneration, they perform distinct and dynamic functions in vivo, depending on their polarization states, pro-inflammatory M1 phenotype and pro-healing M2 phenotype are the two extremes of the phenotypes. In the process of natural wound healing, macrophages are involved in all phases with dynamically changing phenotypes, orchestrating the transition from an inflammatory to regenerative phenotype to guide all other cell types to complete the wound-­ healing process [70, 71]. When macrophages encounter implanted scaffolds, their phenotype would be affected and even regulated by the scaffolds that are necessary when a tissue defect is large beyond the critical size. Therefore, harnessing macrophages by scaffolds is important for facilitating tissue regeneration in situ. To explore the effects of mechanical forces generated by the combination of superparamagnetic scaffolds and magnetic fields on macrophages, macrophage cell line RAW264.7 cells were seeded on nanofibrous scaffolds composed of γ-Fe2O3 nanoparticles (MNP), hydroxyapatite nanoparticles (nHA) and poly lactide (PLA) and subjected to magnetic fields (MF) that was applied alternatively in an interval of 12  h. The magnetically actuated scaffolds did not affect the proliferation of RAW.7, but showed regulatory effects on the phenotype of the macrophages. As shown that the level of Arg1 was upregulated significantly in magnetically actuated scaffolds (mag-S+MF) and that of iNOS was decreased in comparison with that in the other groups. These variations suggested that mag-S+MF guided the macrophages to M2 phenotype and had their M1 phenotype weakened, which was verified by the cytokines profiles, as shown that pro-inflammatory cytokines (IL-1β, TNF-a, and MCP-1) were significantly decreased, and wound healing associated cytokines (IL-4 and IL-10) were significantly increased in mag-S+MF group compared with those in control group (Fig. 1.3). Interestingly, the conditioned medium prepared from macrophages grown in mag-S+MF promoted human umbilical vein endothelial cells (HUVECs) to form more vessel-like tubes, which could be attributed to the enhanced production of VEGF and PDGF of macrophages. The conditioned medium also played positive roles in the osteogenesis of pre-osteoblast cell MC3T3-E1, the medium showed capacity of recruiting and inducing osteogenesis of MC3T3-E1. At the same time, mag-S+MF could inhibit the secretion of MMP-9 and TRAP, the two molecules are characteristic osteoclast marker proteins. In addition, the level of TLR2 and TLR4 for macrophages in mag-S+MF was down-regulated in western blot analysis, and the nuclear NF-κB expression was reduced as well, while the autocrine loop of VEGF/VEGFR2/HIF-1α for macrophages in mag-S+MF group was obviously activated (Fig. 1.4) [72]. These results explained how the magnetically actuated scaffolds enhanced bone tissue regeneration through regulating the phenotype of macrophages, as well as demonstrated that macrophages are mechanosensitive, their phenotype can be regulated by mechanical stimulations generated by the combination of magnetic scaffolds and magnetic fields. A group investigated the effect of frequency of magnetic fields on the phenotype of macrophages. They prepared RGD ligand-bearing superparamagnetic iron oxide

Fig. 1.3  Cytokine profiles of macrophages grown in mag-S or con-S under different MFs for 72 h. Data are means ± SEM. ∗∗p 4000 μm2. On the

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surface of the fibers, GNPs were evaporated with a nominal thickness of 10 nm to give the fibers conductivity. As a consequence, the composite scaffolds promoted cardiac cell organization into elongated and aligned tissues generating a strong contraction force, high contraction rate and low excitation threshold [91]. This deposition strategy is especially suitable be applied to biological materials that are not able to endure complicated and harsh chemical reactions. For example, GNPs were deposited on fibrous decellularized omental matrices to make the decellularized matrices conductive, which is an attractive design for fabricating engineering functional cardiac patches for treating myocardial infarction. Consequently, the cardiac cells growing within the composite scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. The hybrid patches displayed superior function, including a stronger contraction force, lower excitation threshold, and faster calcium transients, as compared to pristine patches [92]. In some cases, GNPs are used with graphene together. A conductive biodegradable scaffold was generated by incorporating graphene oxide gold nanosheets (GO-Au) into a clinically approved natural polymer chitosan (CS). The composite scaffold composed of CS and GO-Au nanosheets displayed two folds increase in electrical conductivity in reference to the chitosan alone scaffold, reaching 0.012 S/m that is 1/10 of that for natural heart tissue. At the same time, the scaffold exhibited excellent porous architecture with desired swelling. In particular, the inclusion of GO-Au reduced the degradation rate of the scaffold because GO-Au sheets hinder the penetration of hydrolytic enzymes inside the polymer matrix that slows down the degradation. In a rat model of MI, the conductive scaffold after 5 weeks of implantation showed a significant improvement in QRS interval, which was associated with enhanced conduction velocity and contractility in the infarct zone by increasing connexin 43 levels [93]. 2.3.2.4  Silicon Nanowires Although silicon nanowires are not well known, they have attractive features of high conductivity and mechanical stiffness, but also biodegradable, and their degradation products are found mainly in the form of Si(OH)4 and are metabolically tolerant in vivo. In one study, n-type SiNWs of 100 nm in diameter and 10 μm in length, and with a ratio of 500 (silane to phosphane) were prepared, the conductivity was as high as150−500  μS/μm, which is much higher than that of cell culture medium (∼1.75 μS/μm) and myocardium (∼0.1 μS/μm). The resulting nanowires were used to improve the differentiation and maturation of human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), because current cardiomyocytes derived from hESCs and hiPSCs retain an immature phenotype, including poorly organized sarcomere structures due to the lack of ability for assembling in a controlled manner and led to compromised, unsynchronized contractions. In this study, a trace amount of electrically conductive silicon nanowires (e-SiNWs) was added in scaffold-free cardiac spheroids to create highly electrically conductive microenvironments within spheroids, leading to synchronized and significantly

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enhanced contraction, as well as significantly more advanced cellular structural and contractile maturation [94]. In a follow-up study, researchers investigated important factors that could affect functions of hiPSC cardiac spheroids cultured with silicon nanowires, including cell number per spheroid and the electrical conductivity of the silicon nanowires. They explained with a semi-quantitative theory that the two factors were competitive in the improvement of 3D cell–cell adhesion and the reduction of oxygen supply to the center of spheroids with the increase of cell number, and the critical role of electrical conductivity of silicon nanowires was confirmed in improving tissue function of hiPSC cardiac spheroids [95]. 2.3.2.5  M  echanistic Exploration of Conductive Scaffolds Enhance Cardiac Tissue Regeneration Several studies have explored and discussed underlying mechanisms of how conductive scaffolds promote the mechanical integrity and electrophysiological function of cardiac myocytes. For example, single-walled carbon nanotubes were incorporated into collagen or GelMA that was used as growth supports for neonatal cardiomyocytes. Researchers demonstrated that the composite hydrogel enhanced cardiomyocyte adhesion and maturation through β1-integrin-mediated signaling pathway. As is known, a variety of intracellular signaling pathways can be initiated by β1-integrin signaling, including FAK, Src, and ILK, and it was found out that CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. In the investigation, a notably higher level of p-FAK was observed in a composite of carbon nanotubes and collagen (CNT-Col) compared to those on collagen alone at all the measured time points, whereas no remarkable changes of p-Src and ILK were noted between two groups, suggesting that FAK was activated and acted as a downstream signaling molecule from β1-integrin. Once FAK is phosphorylated, several downstream signaling kinases can be further activated, including AKT and ERK. Western blotting analysis revealed significant increases in the expression of p-ERK in NRVMs grown on CNT-Col substrates at various developmental phases while no apparent change of p-AKT expression was observed between the two groups, suggesting that ERK was activated by FAK and might be the downstream effector of FAK [96]. It was further demonstrated that the downstream signaling protein RhoA and FAK was responsible for CNT-induced upregulation mechanical junction proteins of NRVMs grown on CNT-GelMA substrates [97]. A recent work compared the effect of mechanical stiffness and nanoscale topography of scaffolds on cell–cell coupling, maturation, and electrical excitability of engineered cardiac tissues. The conductive performance was adjusted by the incorporation of conductive gold nanoparticles or nonconductive silicon nanoparticles. Scaffolds contained silicon nanoparticles. Four different hydrogels were prepared including soft GelMA gel, stiff GelMA gel, GelMA gel containing silicon nanoparticles (nonconductive with nano-topographies and soft), and GelMA gel containing gold nanoparticles (conductive with nano-topographies and stiff). Results highlighted that the influence of nanoscale topography provided by the integrated

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nanomaterials was stronger than the elastic modulus and conductivity on cellular adhesion and retention, as well as on the promotion of cardiac cells maturation, suggesting the prominent role of the nanoscale surface topography of nanocomposite scaffolds in the cardiac cell–cell coupling, maturation, and functionalities of the engineered tissue [98]. These results worth of further consideration though they are not consistent with experimental results from nanofibrous scaffolds containing conductive polymers. To gain insights from different views, we fabricated a conductive hydrogel (PVA/CFs) by blending carbon fibers of 7 μm in diameter and PVA that has a simple molecular structure and forms very soft hydrogels [99]. In this composite hydrogel, there were not obvious nanostructures while the carbon fibers are highly conductive with high tensile strength as well as modulus. In addition, the conductive element was carbon, instead of gold. The carbon fibers brought distinguished conductivity of 0.3 S/m and stiffness of 2.3 MPa to the PVA. It was observed that α5 and β1 integrin were both significantly upregulated in neonatal rat cardiomyocytes grown on PVA/CFs compared with those on PVA control, meanwhile the levels of ILK and p-AKT were elevated correspondingly. In addition, hypoxia-­ inducible factor-1α (HIF-1α) was increased, which was assumed that the cells on PVA/CFs were subjected to a stronger material-support pulling, therefore might experience hypoxia, implying that CFs might provide a microenvironment of promoting angiogenesis in the cells (Fig.  2.3). Noted that these signals are highly

Fig. 2.3  Mechanotransduction signaling is involved in the modulation of CFs with NRCMs. Western blot analysis of the expression of (a) α5 integrin, (b) β1 integrin, (c) ILK, (d) p-AKT, (e) RhoA, and (f) hypoxia-inducible factor 1-α in NRCMs grown on PVA/CFs or PVA alone for 5 days. Data are represented as mean ± standard deviation (SD); ∗p