Chapter 9 - Tissue Engineering Using Magnetite Nanoparticles

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The major advantage of magnetic manipulation is “remote control.” Magnetic labeling of cells with magnetic nanoparticles enables the manipulation of cells and also the control of cell functions by applying an external magnetic field. “Functional” magnetite nanoparticles were developed for cell manipulation using magnetic force, and the magnetite nanoparticles were applied to tissue-engineering processes, which are designated as magnetic force-based tissue engineering (Mag-TE). This chapter reviews recent progress in Mag-TE techniques, and the principles and utilities of the applications are discussed. This review covers three topics of magnetic cell manipulation using magnetite nanoparticles, including a magnetic force-based gene transfer technique (magnetofection), magnetic cell patterning using functional magnetite nanoparticles and micro-patterned magnetic field gradient concentrators, and finally applications for fabrication of tissue-like constructs in skin, liver, and muscle tissue engineering.

Introduction

Since magnetic particles have “magnetic” properties that are not seen in other materials, they have been applied to various medical techniques such as cell separation,1 drug or cell targeting,2, 3 magnetic resonance imaging (MRI),4 and hyperthermia.5 The magnetic particles most frequently used for cell separation are ferrites with a general composition of MFe2O3 (where M represents a divalent metal cation, such as Ni, Co, Mg, or Zn, and includes magnetite Fe3O4) and maghemite Fe2O3. For medical applications, the most important feature is nontoxicity of magnetic particles. Based on this criterion, magnetite nanoparticles have been mainly and extensively studied and are being used in an increasing number of biological and medical applications.6, 7

In order to add an affinity and targeting ability for cells, the concepts involved in drug delivery systems were applied to magnetite nanoparticles and functionalized magnetite nanoparticles were developed. Three types of functionalized magnetite nanoparticles are illustrated in Fig. 1. Magnetite cationic liposomes (MCLs), in which 10 nm magnetite nanoparticles are encapsulated into 200 nm cationic liposomes, were developed to improve the accumulation of magnetite nanoparticles in target cells through electrostatic interactions between MCLs and the cell membrane.8 Additionally, among cell-manipulating techniques, control of cell adhesion is one of the most important issues. To promote cell attachment, MCLs were modified with an RGD (Arg-Gly-Asp) peptide, an integrin recognition motif found in fibronectin,9, 10 and a well-studied cell adhesion peptide, designated RGD-MCLs.11 The average particle size of RGD-MCLs was 240 nm, and this size was similar to that of the MCLs. As an opposite concept, development of functionalized magnetite nanoparticles possessing the ability to resist cell attachment enables spatial control of cell adhesion onto cultural substrates. One of the most useful polymers to repel proteins is poly(ethylene glycol) (PEG). Surface modification with PEG leads to a significant reduction in the nonspecific interaction of biological molecules with the surface due to its high degree of hydrophilicity and chain flexibility.12, 13 Thus, 220 nm PEG-conjugated magnetite nanoparticles (PEG-Mags) were developed for spatial control of cell adhesion.14

Tissue engineering applies the principles of biology and engineering to the development of functional substitutes for damaged tissue.15 There has been growing enthusiasm for tissue engineering, and this new technology has been a promising approach for overcoming the organ transplantation crisis resulting from donor organ shortage. Tissue engineering comprises the following processes (Fig. 2): (1) autologous cells isolated from healthy tissues or stem cells including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells16 are expanded to the required cell number; (2) genes of interest may be transferred into cells to enhance or modify cellular functions; (3) three-dimensional (3D) tissue-like structures are constructed, allowing 3D cell culture; in this step, if necessary, cells are cocultured with various cell types and/or patterned to mimic natural tissue structures; and (4) the cultured 3D constructs are transplanted into patients. Although overall technology of these processes in tissue engineering has been established, there is still plenty of room for improvement in each process.

Procedures to manipulate and remotely control cellular behavior can provide a powerful tool for tissue engineering. Magnetic manipulation offers such a tool, and the major advantage of magnetic manipulation is that it allows action from a distance. Dobson et al.17, 18 reported magnetic actuation for the mechanical conditioning of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. They used a range of magnetic particle sizes from 130 nm up to 4 μm and showed that the technique was effective for stimulation of intracellular calcium storage, membrane potential change, and upregulation of genes related to bone and cartilage formation in MSCs. In 2006, Ingber et al.19 developed a magnetic force-based scaffold construction procedure. They used magnetic fields to position thrombin-coated magnetic nanoparticles in two-dimensional (2D) hexagonal arrays. The particles acted as nucleation sites for the ordered growth of fibrin, creating an ordered fibrin gel scaffold for endothelial cells. Moreover, magnetic manipulation presents distinct advantages for in vivo applications. In 2007, Wilhelm et al.20 demonstrated that endothelial progenitor cells, which may facilitate angiogenesis and revascularization in ischemic sites, can be remotely guided both in vitro and in vivo by applying a magnetic force.

From the viewpoint of bioprocess engineering, development of a methodology for physical manipulation of target cells is essential for tissue engineering in the next generation. A magnetic force was selected as a tool for physical manipulation, and target cells were manipulated using the functionalized magnetite nanoparticles. Thus, a novel cell-manipulating technology was developed using functionalized magnetite nanoparticles and magnetic force, designated as magnetic force-based tissue engineering (Mag-TE). This chapter focuses on Mag-TE techniques that have been applied to tissue-engineering processes: (1) gene transfer (magnetofection); (2) cell patterning; and (3) fabrication of tissue-like constructs.

Section snippets

Magnetofection

Growth factors stimulate cells for proliferation, differentiation, survival, and/or extracellular matrix (ECM) synthesis, and they are therefore a key element of tissue engineering. However, many problems arise with the use of growth factors, including the transient effect of these proteins due to their relatively short biological half-lives and the difficulty in delivery to a specific injured site. Thus, gene delivery technology has become a crucial issue in recent years for establishing

Magnetic Patterning of Cell

Tissue engineering aims to create functional tissues using cells, growth factors, and biomaterials. In addition, if tissue-engineered architectures are completely similar to organs in vivo, tissue-engineered equivalents can be used for studies in cell biology or for evaluating the effects of drugs and toxins, which can lead to a reduction in the use of research animals. However, it is difficult to construct functional organs because tissue-engineered architectures are not completely similar to

Construction of 3D Tissue-Like Structures

Conventionally, tissue engineering has been based on the seeding of cells onto 3D biodegradable scaffolds to reconstruct their native structure. Therefore, most efforts in tissue engineering may have been focusing on the scaffold design. The use of biodegradable scaffolds, however, poses problems such as insufficient cell migration into the scaffolds and inflammatory reaction due to the biodegradation of the scaffolds. Especially in muscle tissue engineering, because cell–cell interactions are

Conclusion

This chapter highlighted magnetofection, magnetic patterning of cells, and construction of 3D tissue-like structures. Among them, Mag-TE for constructing 3D structures has been extensively studied, and various kinds of other tissues such as retinal pigment epithelial cell sheets,102 MSC sheets,44 and cardiomyocyte sheets,46 have been already generated. Tubular structures consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts have also been created.43 In this

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