Magnetic‐Based Strategies for Regenerative Medicine and Tissue Engineering

The fabrication of biological substitutes to repair, replace, or enhance tissue‐ and organ‐level functions is a long‐sought goal of tissue engineering (TE). However, the clinical translation of TE is hindered by several challenges, including the lack of suitable mechanical, chemical, and biological properties in one biomaterial, and the inability to generate large, vascularized tissues with a complex structure of native tissues. Over the past decade, a new generation of “smart” materials has revolutionized the conventional medical field, transforming TE into a more accurate and sophisticated concept. At the vanguard of scientific development, magnetic nanoparticles (MNPs) have garnered extensive attention owing to their significant potential in various biomedical applications owing to their inherent properties such as biocompatibility and rapid remote response to magnetic fields. Therefore, to develop functional tissue replacements, magnetic force‐based TE (Mag‐TE) has emerged as an alternative to conventional TE strategies, allowing for the fabrication and real‐time monitoring of tissues engineered in vitro. This review addresses the recent studies on the use of MNPs for TE, emphasizing the in vitro, in vivo, and clinical applications. Future perspectives of Mag‐TE in the fields of TE and regenerative medicine are also discussed.


Introduction
Owing to the complexity of native tissues, the in vitro fabrication of completely functional tissues that closely mimic the in vivo environment remains a significant challenge in TE. [1] Cells are typically cultured on tissue culture polystyrene dishes and harvested using the proteolytic action of trypsin or the chelation of Ca 2+ and Mg 2+ ions with ethylenediaminetetraacetic acid (EDTA) to disrupt cell-cell junctions with a substrate.However, the interactions between cells and components of the extracellular matrix (ECM), such as proteins and integrins, are crucial for tissue development and maintenance. [2]The introduction of stimuli-responsive materials has allowed the development of new methodologies to reverse cell attachment/detachment, avoiding DOI: 10.1002/adhm.202300605damage to cell membranes and the ECM caused by nonspecific proteases. [3]In recent decades, magnetic responsive systems have been explored in TE and regenerative medicine to achieve favorable outcomes in drug delivery, cell therapy, and in vivo monitoring.Magnetically labeled cells have been developed to fabricate tissue replacements with fine control over cell positioning and to support tissue regeneration. [4,5]MNP labeled cells can be directed to a specific location using a magnetic field to prompt the repair of the tissue or restore its function. [6]he magnetic characteristics of these tissue substitutes can also offer insights into the accurate positioning and functionality of cells and materials over time by monitoring bioengineered materials.Several preclinical in vitro and in vivo studies have successfully demonstrated magnetic-based cell therapy and TE and its monitoring using magnetic resonance imaging (MRI) for the regeneration or replacement of diseased or damaged tissue. [7]However, clinical trials involving the application of magnetic fields are still restricted to monitoring purposes.This review highlights recent advances in magnetic-responsive systems for regenerative medicine, including TE monitoring, magnetic cell guidance, and tissue regeneration (Figure 1).

TE: Different Approaches to Create and Repair Tissues
TE assists in restoring the functionality of injured tissues and/or organs resulting from trauma, aging, or disease. [8]The replication of the complex architectural features of native organs and tissues is of paramount importance for guaranteeing the proper functionality of engineered tissues.Traditional TE approaches use a "top-down" strategy, in which cells are seeded onto a scaffold with biocompatible and biodegradable features. [9]In this case, cells are likely to populate the scaffold and develop their own ECM and suitable microarchitecture.Mechanical stimulation, perfusion, and incorporation of biomolecules, such as growth factors and signaling molecules, have been used to facilitate the formation of these structures. [10]Commonly used techniques for fabricating top-down scaffolds include freeze-drying, [11] solvent casting, [12] electrospinning, [13] and supercritical fluids. [14]Thin or avascular tissues, including skin, cartilage, and bladder, have been successfully engineered using "top-down" approaches. [15]igure 1.Representative illustration of the major fields of action of magnetic TE: monitoring TE, magnetic guidance and development of tissue constructs using cell patterning, and scaffold-based and scaffold-free strategies.
However, the fabrication of larger and more complex tissuelike constructs remains challenging.Top-down approaches often encounter challenges when attempting to reconstruct the intricate microstructural characteristics of tissues, resulting in nonfunctional constructs.Inspired by the building blocks that compose the complex hierarchy of tissues and that regulate their entire function, "bottom-up" approaches have also been explored in the engineering and assembly of microscale tissue building blocks with a specific microarchitecture to engineer larger tissue constructs. [16]Several features such as the shape and composition of individual blocks can be easily controlled, thereby enabling the development of versatile tissue structures. [17]Cellencapsulating microscale hydrogels (microgels), self-assembled cellular aggregates, cell sheet (CS) generation, and direct printing of cells have recently been employed to fabricate these tissue building blocks. [18]

TE Via External Stimulus
The new generation of "smart" biomaterials is at the forefront of innovation in the medical field, revolutionizing conventional areas of TE, drug delivery, and manufacturing of medical devices.Biomaterials can be engineered to modulate their chemical and mechanical properties in response to external signals and/or the surrounding environment. [19]Temperature, pH, redox state, light, and magnetic fields are the most promising stimuli explored in the field of "smart" biomedicine.Precise responsiveness to stimuli and the capability to react to endogenous cues inherent in living systems offer opportunities to elevate the conventional field of TE into a more precise and sophisticated paradigm.
However, the translation of these new materials into clinical practice remains challenging.Thermoresponsive polymers have been extensively explored in the biomedical field, permitting engineering of CSs, in situ drug delivery, and three-dimensional (3D) printing under physiological conditions. [20]In particular, poly(N-isopropylacrylamide) has been employed in the fabrication of thermoresponsive surfaces for TE purposes, acting as a substrate for thermoresponsive cell culture dishes.In contrast to traditional strategies, thermoresponsive surfaces allow the recovery of unharmed and interconnected CSs without destroying the cell junctions, [21] which can be achieved by reducing the temperature below the lower critical solution temperature (LCST), which triggers cell detachment. [22]However, these substrates are mainly constrained by the elevated costs and diminished mechanical strength of the resultant CSs.Furthermore, they are hindered by abnormalities associated with physiological alterations in the cellular microenvironment. [23]esponsive systems that exploit the different pH values within the human body to induce a more targeted response have also been explored.Owing to the acidic extracellular pH of tumor cells, pH-responsive systems have received considerable attention as drug delivery platforms, particularly for anticancer agents.They are also being investigated for oral drug administration, exploiting pH variations in the gastrointestinal tract. [24]With regard to TE applications, cell-based tissue constructs were successfully obtained by detaching the CS by decreasing the pH of the cell culture. [25]owever, changes in pH may be detrimental to more sensitive cells, and consequently, this type of system is poorly explored for TE application.Electrical stimulation is a recently developed tool in TE and regenerative medicine that acts on the morphology, orientation, migration, and phenotype of several cell types.These systems have shown promising results for wound healing and bone regeneration in both animal experiments and clinical treatments. [20]For the fabrication of tissues in vitro, polyelectrolyte-modified surfaces have been used for CS fabrication based on the mechanism of absorption and desorption of oppositely charged surfaces.With the aid of a positive potential, the surface monolayer desorbs and the cells lose their attachment points, enabling CS harvesting. [26]Recurrent charge accumulation during the application of an electric field affects cell behavior and can induce a nonhomogenous distribution of the cells, limiting their use in TE applications. [27]ght-responsive smart biomaterials are also an attractive strategy for TE because they are involved in the fabrication of complex scaffolds, control of cellular behavior, release of key factors, and tissue regeneration.The application of a light stimulus confers some advantages over alternative strategies as it enables the instantaneous imposition and precise delivery of predetermined doses, affording spatial and temporal control using less invasive techniques. [28,29]Light-responsive composite coatings have shown promising results in regenerative medicine, allowing for the control of cell attachment/detachment based on wettability changes. [30]This concept inspired the fabrication of CSs in which a completely cell-based membrane could be recovered after UV irradiation at a cell-safe wavelength of 365 nm using a coated surface as the substrate. [31]Several biosynthetic soft tissues have been engineered using photopolymerization. [32]Briefly, the prepolymer is injected into the damaged site, enabling tissue reconstruction in situ after light exposure. [33]Nevertheless, the scientific community has highlighted several concerns regarding the use of photopolymerization in TE.The energy emitted by the polymerizing light, heat, and radical species created upon polymerization and the potential toxicity of photoinitiators and monomers may harm the surrounding tissues and/or encapsulated molecules. [34]

Magnetic TE
The remarkable features of magnetically responsive materials have prompted their use in the biomedical field, particularly in TE.However, the biocompatibility and long-term stability of materials such as MNPs are still major concerns. [35]Despite being considered biologically safe, the extensive and exponential use of MNPs in various biomedical applications in recent decades has prompted extensive research on their cytotoxicity and biological fate.Initial findings indicate that MNPs can be deemed biologically safe because of the effective regulation of iron cell homeostasis through processes such as uptake, excretion, and storage, leading to the efficient clearance of excess iron from the body.Notably, in vivo assays conducted in rat models demonstrated that these iron-based particles do not induce oxidative stress or cause long-term alterations in the levels of liver enzymes, which serve as indicators of biosafety. [36]However, the safety of these materials is significantly influenced by several parameters, including particle size, shape, functionalization, and concentration.Collectively, these factors affect the colloidal stability of MNPs, consequently influencing their interaction with cells. [37]o facilitate clinical translation, the scientific community should establish stringent and well-defined guidelines encompassing reproducible procedures for developing stable and biocompatible formulations characterized by appropriate and consistent particle sizes, designs, and surface modifications.
Superparamagnetic NPs have gained significant attention in the field of regenerative medicine because of their implementation in biological systems.These types of MNPs, which are characterized by their ability to exhibit no residual magnetization upon the removal of an applied magnetic field, possess distinct advantages.This property prevents nanoparticle aggregation and allows for rapid redispersion once the magnetic field is removed.Moreover, they offer advantages in terms of tissue penetration and invasiveness, as biological tissues have a lower absorption capacity for magnetic fields than for other types of stimuli such as electric fields.39][40] Figure 1 illustrates the potential of MNPs in the biomedical field.Starting with more conventional strategies, MNPs allow the use of a minimally invasive methodology to perform cell separation, drug and cell targeting, and delivery of small molecules.MNPs have also played a crucial role in monitoring using MRI and in the treatment of challenging diseases such as cancer using magnetic hyperthermia.More advanced strategies employ MNPs to engineer functional tissue substitutes. [41]The major achievements in the fabrication of magnetized tissues are presented in the following sections, highlighting key approaches for tissue development and their potential applications.

Monitoring of TE Processes
In regenerative medicine, replacement tissues can be produced a priori and transplanted or assembled de novo at the site of injury.In both scenarios, the implanted tissue constructs must be monitored over time to evaluate tissue development and functionality.Therefore, the development of suitable methodologies for tissue monitoring is critical for the application of tissue substitutes.To achieve appropriate tissue regeneration, the structure, composition, and strength of the developing tissues must be investigated throughout the process. [42]Conventionally, tissue growth is monitored using optical and fluorescence microscopy techniques.However, even more sophisticated methodologies, such as multiphoton imaging, possess a restricted depth of field.Although techniques such as microcomputed tomography can solve tissue penetration problems, these methods use ionizing radiation and require dense tissue for contrast. [43]Exposure to ionizing radiation, even at low frequencies, poses a significant health hazard.The underlying mechanisms include the elicitation of reactive oxygen species, modulation of gene expression, and induction of DNA damage through both epigenetic and genetic processes. [44]In contrast, MRI offers a promising alternative imaging modality as it uses nonionizing radiation and a paramagnetic agent, that is, MNPs, and shows promising results for in vivo imaging because of its optimal tissue penetration and 3D spatial resolution.MRI is a highly sensitive method that allows the visualization of structural and functional changes, even in biological organisms.As tissues undergo development, degeneration, or regeneration throughout the regeneration process, the local tissue environment, particularly water content (the origin of the magnetic resonance (MR) signal), undergoes alterations.These variations are depicted in MR images as localized discrepancies in the tissue water quantity and NMR relaxation times. [45]he heart, brain, kidney, bone, cartilage, pancreas, tendons, and ligaments are the main engineered tissues that have already been assessed using MRI.Until now, the diffusion of oxygen and nutrients, cellular metabolism, and ECM development have been the key parameters typically analyzed using MRI in engineered tissues.However, other important features must be investigated, depending on the target tissue.For example, constructs aimed at cartilage regeneration require a combination of mechanical strength, viscoelastic properties, and the synthesis of collagen II and proteoglycans, whereas bone requires the synthesis of bone matrix, mineralization, angiogenesis, and high loading mechanical strength. [45]Therefore, MRI is currently the most feasible monitoring methodology, permitting the scanning of any type of tissue and analysis of a large set of parameters in different scenarios, such as diagnosis, patient treatment, and tissue regeneration.The magnetic field has opened new tools for in vivo imaging, overcoming the main problems of conventional methodologies.

Magnetic Guidance
Conventional delivery systems often lack tissue and cell specificity and face significant challenges in overcoming inherent physiological barriers. [46]To overcome these limitations, electromagnetic devices and MNPs have been integrated to establish magnetic guidance systems in the context of TE, simultaneously enabling the guidance and tracking of drugs, cells, and/or biomolecules.
As stated in the previous section, MRI is considered the most efficient method for the feedback control of targeted biomolecules, enabling precise real-time tracking of magnetic nano/microdevices.In regenerative medicine, the use of labeled MNPs enables the targeted delivery of isolated or aggregated cells to specific tissues and organs while providing real-time monitoring capabilities.
However, because the MRI image reflects signals from blood, muscles, and tissues to depict the body/organ anatomy, its application in drug delivery monitoring may be compromised by background signals from the host tissue.To overcome this issue, magnetic particle imaging (MPI) has emerged as a novel, noninvasive, and radiation-free imaging technique that can quantify MNP tracers.This strategy allows the creation of fully 3D images of the injected tracer particles without exhibiting a background of soft tissues, thus overcoming the challenges of MRI. [47]MPI scanners offer a remarkable capacity to achieve rapid imaging with high sensitivity and deliver millimeter-scale resolution, and their outstanding potential has the capability to instigate a transformative impact in the field of biomedical imaging.
Focusing on the sustained release of bioactive compounds, Li et al. demonstrated that the controlled release of drugs can be achieved by remotely applying a low-frequency oscillating magnetic field.This behavior has been demonstrated in magnetic hydrogels containing dextran and insulin.Remote release of the drugs was achieved owing to temperature changes in the magnetic hydrogel, which was composed of a thermoresponsive polymer, in the presence of a magnetic field. [48]Upon increasing the temperature above the LCST, a fraction of the magnetic hydrogel collapsed, resulting in the release of the encapsulated drugs.The presence of an alternating magnetic field led to significant differences in the swelling ratio of the magnetic hydrogels.Upon activation in the "on" state, the magnetic hydrogel exhibited a diminished swelling ratio, which subsequently increased and nearly returned to its original state upon switching to the "off" state. [44]his phenomenon is attributed to alterations in the porosity or pore size of the magnetic hydrogel under the switching "on-off" mode.This strategy has already been explored for neuromodulation by combining genetic targeting of a magnetoreceptor with remote magnetic stimulation in a noninvasive manner. [45]The magnetogenetic control of neuronal activity might be dependent on the direction of the magnetic field and exhibit on-and offresponse patterns when an external magnetic field is applied.Compared to conventional methodologies such as optogenetics (a method that allows the activation or inhibition of light-sensitive ion channels commonly used for the spatial and temporal manipulation of neurons), magnetogenetics (a technique that employs a magnetic field to remotely regulate cell activity) offers several advantages, including noninvasiveness, deep penetration, longterm continuous dosing, unlimited accessibility, and spatial uniformity.
Direct cell therapy and simple approaches, such as injection of magnetized cells, have been explored to promote the accumulation of cells at the target site.For example, Polyak et al. revealed that re-endothelialization of the carotid artery was attained in the left ventricular cavity of rats by injecting previously magnetized aortic endothelial cells. [49]However, more sophisticated on-off remote systems have also been engineered. [50]Xia et al. employed this system for the controlled release of stem cells using composite scaffolds.Diverse release profiles were obtained by modulating the intensity and frequency of the external magnetic field, as well as the number of magnetic cycles.Later, Wang et al. demonstrated that the release rate of both hydrophobic and hydrophilic drugs could be increased by applying an alternating magnetic field. [51]n contrast to conventional approaches, magnetically guided methodologies enable finer, more targeted, and more efficient procedures.Drugs, biomolecules, and cells can be tracked and guided with high precision.

Cell-Patterning
Magnetic-force-based cell patterning platforms have been developed to merge magnetic forces with micropatterning technologies for TE purposes.Owing to the high accuracy of these platforms, single-cell arrays can be developed to study individual cell behavior and morphological alterations, with potential applications in cell diagnosis and studies of cell-cell interactions. [52]Devices composed of spaced micropillars are among the most explored configurations for creating arrays of single adherent cells.For example, Ino et al. fabricated a magnetic holder device with an array of pillars that acted as substrates to attract magnetized cells. [52]This magnetic device was used to investigate cell function and behavior during angiogenesis, as shown in Figure 2A.For this purpose, magnetic-labeled cells were rearranged in accordance with the micropatterned device, promoting the formation of cell clusters in adjacent spots and, therefore, the creation of branched structures.Cell-cell interactions are crucial for angiogenesis.
More ambitious systems have also been proposed, including the 3D fabrication of magnetic-patterning systems. [53]Goranov et al. developed a 3D magnetic scaffold for fabricating vascularized tissues.The proposed scaffold was composed of separate arrangements of vascular and osteoprogenitor cells established on opposite sides of the scaffold fibers, showing promising features for the development of materials with angiogenic potential. [54]ochi et al. also explored such 3D arrays to analyze the invasive capacity of BALB/3T3 cells, which were used as a cancer model cell line (Figure 2B).The 3D cell patterning was conducted using an external magnetic force and a pin holder, facilitating the arrangement of magnetically labeled cells on a collagen gel-coated surface.The developed 3D cell culture array system is promising for evaluating the impact of pharmacological and/or microenvironmental factors on tumor cell invasion. [52]hatley et al. employed an analogous approach for the fabrication of 3D cell-based structures using endothelial cell spheroids as the basic units.After the cellular uptake of the MNPs and the formation of magnetized spheroids, they were directed toward a preestablished magnetic template through the attractive magnetic forces between the magnetized spheroids and the magnetic template. [55]The magnetic spheroids began to fuse over time, and 3D multicellular tissue constructs were obtained by removing the template (Figure 2C).This strategy allows the patterning of cell spheroids in accordance with the desired magnetic patterns, thus exhibiting promising potential for the scalable fabrication of tissue structures characterized by a high degree of complexity.Stratified tissue structures have also been designed by combining cell patterning with magnetic force.Akiyama et al. described the fabrication of multilayered CSs using a magnetic concentrator.Briefly, a layer of C2C12 cells (a myoblast cell line) was formed, and line patterning of human umbilical vein endothelial cells (HUVECs) was developed on top of the previously formed cell monolayer. [56][58] The 3D arrangements were controlled by a magnetic concentrator and magnetization of the material, allowing the fabrication of fine and distinct biomaterials such as cell aggregates, fibers, spheroids, and CSs.

Scaffold-Based TE Strategy
Conventional scaffold-based approaches rely on the use of "templates" that support the attachment and proliferation of living cells to form 3D tissues. [59]In recent decades, the feasibility of custom-designed materials has propelled the application of scaffolds in TE.An ideal scaffold should exhibit a wide array of cues, including chemical, biochemical, and biophysical factors capable of governing and facilitating precise cellular and tissue-level processes, such as chemical, biochemical, and biophysical processes, and can control and promote specific events at the cellular and tissue levels. [60]3D scaffolds are expected to protect cells from damage associated with external factors while providing a biomimetic environment for cells, with the delivery of growth and differentiation factors that lead to tissue formation. [61]everal material types, such as natural, [62] synthetic, [63] semisynthetic, and hybrid materials, [64] have been employed to fabricate scaffolds for TE (Figure 3A).Despite the many advantages offered by materials derived from natural sources, their application has been limited by possible immunogenicity, purification issues, and difficulties in controlling their material properties and performance.Conversely, synthetic materials are easily reproducible and programmable; however, their insufficient A) The patterning of single cells was achieved using a magnetized pin holder device, promoting the formation of cord-like structures, which are crucial during angiogenesis.B) Similar devices were used as a cancer model to investigate the invasive capacity of BALB/3T3 cells.C) Three-dimensional (3D) tissue constructs were also fabricated using this strategy, where magnetic spheroids formed à priori were guided to a prefabricated magnetic template.Finally, the spheroids fused and a 3D tissue with the geometry of the template was formed.Reproduced (adapted) with permission. [50]Copyright 2009, Wiley-VCH GmbH.Reproduced with permission. [52]Copyright 2009, Royal Society of Chemistry.Reproduced with permission. [53]Copyright 2014, Wiley-VCH GmbH.
biological recognition limits their application as tissue regeneration matrices. [65]Alternatively, hybrid materials combine the performance and properties of synthetic materials with biomolecular cues capable of directly interacting with cells. [66]he scaffolds must replicate the ECM of native tissues to guide the adhesion, migration, and proliferation of living cells. [67]Several ECM signals, such as fibronectin (FN), laminin, and integrin molecules, which are key regulators of cell adhesion and migration, have been conjugated to scaffolds to create synthetic ECM analogs. [68]However, the use of scaffolds in TE is associated with certain limitations.In porous 3D scaffolds, there is a tendency for cell distribution to be nonuniform, with a low initial density, particularly in the case of larger constructs. [69]To overcome this problem, researchers have engineered hydrogels in the presence of cells that not only enable homogenous distribution, but also lead to higher cellular densities. [70]Hydrogel microcapsules are among the most explored microscale hydrogels with a focus on cell-encapsulation strategies.This approach allows for the transplantation of nonautologous cells that are protected from the immune system of the host owing to their encapsulation in a semipermeable hydrogel membrane. [71]This membrane enables the diffusion of nutrients and cellular metabolic products, while effectively excluding antibodies and immune cells.Multishaped complex structures with clinical relevance have been engineered by the assembly of these cell encapsulation systems in accordance with the concept of modular TE. [72] Considering the intricate architecture of native tissues, nanoand microstructured materials were incorporated into the hydrogel network to mimic its nonhomogenous structure. [73]Because of the remote actuation of MNPs, such materials have been increasingly explored in the biomedical field, including the manipulation of the MNP distribution within the 3D space of hydrogel networks, thereby enabling the controlled design of anisotropic magnetically responsive scaffolding materials.Such smart hydrogels have been introduced into the biomedical field for several applications such as drug delivery, bioseparation, biosensors, and TE.Moreover, magneto/mechanical stimuli can be used to regulate the growth, migration, proliferation, and differentiation of cells enclosed within magnetic hydrogels, directing them toward specific lineages.This enables the fabrication of cell-laden .SEM images of porous hydrogels, where bone mesenchymal stem cells (BMSCs) adhered and aggregated in the pores.A small number of cells adhered to the porous surface of the control (bottom).C) Micrographs of a hybrid hydrogel containing collagen type II, HA, polyethylene glycol (PEG), and MNPs (top).These materials were explored for cartilage TE, where the collagen was extracted from sheep femur condyle with potential calcification (bottom).D) Micropatterned platform for the high throughput fabrication of microtissues with complex geometry (on left); these magnetic hydrogels can be used to produce larger structures using magnetic-guided assembly (right).E-i) Engineering of macro-scale tissues by random or shape-directed assembly of magnetic microcryogels under magnetic force, and ii) photographs of circle and clover magnetic microcryogels and their accurate assembly.Even at the macroscale, the tissue constructs remained viable (live/dead images in Figure 4Biii).Reproduced with permission. [55]Copyright 2016, Wiley-VCH GmbH.Reproduced with permission. [76]Copyright 2018, American Chemical Society.Reproduced with permission. [77]Copyright 2015, American Chemical Society.Reproduced with permission. [81]Copyright 2014, Royal Society of Chemistry.
constructs exhibiting precisely ordered characteristics, and closely replicates the intricate architecture of native tissues.This behavior is mainly attributed to the physical, biochemical, and mechanical alterations of the milieu surrounding the cells and tissues in response to external magnetic stimuli. [74]Magnetic hydrogels have shown promising results in recreating the native structures of neural, heart, skin, cartilage, and bone tissues. [48]or example, the incorporation of Fe 3 O 4 nanoparticles and curcumin into N-isopropylacrylamide-methacrylic acid hydrogels reduced doxorubicin-induced cardiac toxicity, thereby exhibiting a cardioprotective capability. [75]The regeneration of muscle tissues by conjugating ferrogel scaffolds and alginate has also been reported.Using magnetic stimulation (5 min at 1 Hz every 12 h), the biomaterial was mechanically activated and regeneration of the injured muscle tissue was promoted. [76]uang et al. demonstrated the capability of PEG magnetic hydrogels to induce fibroblast alignment, which is crucial for wound healing.The presence of a magnetic field in collagen hydrogels containing MNPs triggered the alignment of collagen fibers.Neurons embedded within a 3D-aligned magnetic hydrogel matrix, as compared with those without fiber alignment, exhibited robust cell viability and enhanced elongation and directional growth.These findings suggest that 3D-aligned magnetic hydrogels hold significant promise for promoting directed neuronal regeneration. [77]he major achievements in magnetic TE have focused on engineering the cartilage and bone tissue.Huang et al. developed a magnetic nanocomposite hydrogel composed of PVA, HA, and Fe 2 O 3 MNPs for use in cartilage TE. [78] The hydrogel regulated the differentiation of BMSCs (Figure 3B).Moreover, the introduction of MNPs improved the mechanical performance of the hydrogel and promoted the development of microporous structures on the hydrogel surface, favoring both cell adhesion and proliferation (Figure 3C). [79]Scaffolds composed of MNPs, collagen II, HA, and PEG exhibited a similar microstructure and chemistry to hyaline cartilage tissue, while ensuring the biocompatibility of the proposed device. [80]This behavior has been verified using other materials, including hydrogels composed of fibrin agarose, MNPs, and hyaline chondrocytes.The resulting artificial tissues exhibited a stronger and more stable mechanical response with the introduction of MNPs, promising in vitro cytocompatibility and correct deposition of collagen type II.
Magnetic hydrogel composites have also been developed for bone TE.Farzaneh et al. proposed a polyacrylic acid hydrogel with cobalt ferrite nanoparticles (CoFe 2 O 4 ) as the magnetic agent. [81]he synergistic effect between the magnetic scaffold and the external magnetic field prompted the osteogenic differentiation of human dental pulp stem cells, leading to elevated alkaline phosphatase activity and enhanced mineral deposition.This approach holds considerable promise as a viable alternative to chemical stimulants for the promotion of osteogenesis.
Henstock et al. demonstrated that collagen hydrogels encapsulating MNPs, bone morphogenetic protein-2 (BMP-2), and human mesenchymal stem cells (MSCs) can stimulate the mineralization of artificial tissues. [82]agnetic hydrogels have also been designed at the microscale to resemble functional living tissue units.The manipulation of microtissues, such as regular medium exchange, can pose a relative challenge to their integrity (Figure 3D,E).To overcome this is-sue, Liu et al. proposed the fabrication of on-chip magnetic hydrogels at the microscale, specifically, microcryogels (Figure 3E). [83]he use of magnetic microcryogels enabled the convenient manipulation of microtissues, mitigating the risk of cell loss during medium exchange, ensuring microtissue integrity throughout long-term culture and facilitating the magnetic purification of the desired functional units.

Scaffold-Free TE Strategy
Despite tremendous efforts in hydrogel engineering for TE applications, several concerns, including scaffold choice, immunogenicity, degradation rate, toxicity of degradation products, host inflammatory responses, fibrous tissue formation, and mechanical mismatch with the surrounding tissue, can collectively affect the performance of engineered tissue constructs, potentially compromising their biological functionality. [72]Additionally, scaffolds are usually linked with decreased cell-to-cell connections and incorrect ECM deposition and alignment, which compromises the target function of the fabricated tissues. [84,85]Moreover, scaffold-based approaches may not fully replicate the intricate, well-organized, and hierarchical architectures exhibited by native tissues and organs.Flat tissues, tubular structures, hollow and nanotubular viscous organs, and complex solid organs exhibit unique features that challenge conventional TE. [86] Therefore, materials science, matrix biology, and TE have been progressively integrated to develop a novel class of biomimetic materials that closely emulate crucial aspects of natural tissues in terms of their physical and chemical properties.To create completely functional tissues, scaffold-free TE has emerged as a powerful strategy to recapitulate native tissues by exploiting the ability of cells to synthesize the entire tissue matrix without the use of exogenous scaffolds (Figure 4A). [87,88]Scaffold-free TE enables the formation of complex tissues with 3D architectures and intensive cell-cell interactions while guaranteeing the correct deposition of the ECM.Due to the high cell density of engineered tissues, this methodology facilitates rapid tissue formation and accelerates tissue maturation. [84]onsidering the heterogeneity of almost all tissues and organs, it is critical to establish a heterogeneous cell culture system for recreating native tissue/organ-like 3D constructs. [89]Monoculture, heterotypic tissues, prevascularization, and specific and complex tissue assemblies are among the diverse topics that have been extensively investigated in TE.Recent advances in the development of cell-based tissue constructs are explored in the following sections, highlighting current methodologies and major outcomes.Although scaffold-free approaches predict that the final biological product ready for implantation should be scaffoldfree, some scaffold-free strategies may accommodate biomaterials with specific cell functions in addition to serving as anchors for tissue growth.The list of scaffold-free systems discussed herein is categorized as cell-based biomaterial approaches because biomaterials may be used to prompt and help orchestrate the specific functions of natural tissues.

Spheroids and Multishape Microtissues:
Most native mammalian tissues exhibit a 3D and highly compact organotypic cellular organization. [90,91]Owing to their architectural and organizational resemblance with native tissue structures concerning .Fabrication of cell-based tissue constructs using scaffold-free approaches.A) Image illustrating the scaffold-free TE strategy, where magnetized cells are used as building blocks for the fabrication of cell-based tissue constructs, namely, spheroids, CSs, and multishaped tissues.B) Production of magnetic spheroids using Mag-TE.i) By using magnetic spheroids as tissue units in the presence of a magnetic pin, the fusion of spheroids into a single tissue construct was accelerated and the union of the spheroids was achieved after 24 h; ii) sequential tissue fusion of vascular tissue spheroids embedded in collagen type I hydrogel, and iii) magnetiferritin nanoparticles in magnetic cellular spheroids.Magnetic-labeled spheroids were assembled onto a ring magnet.The presence of a strong magnet was crucial for the spheroid fusion in the desired shape.The inner diameter contraction was visualized in the presence of a weak magnet.C) Fabrication of CSs using Mag-TE.i) Transplantation of an MSC sheet (single layer) to the cranial bone of nude rats using an electromagnet (on bottom); ii) simple magnetic harvesting process that allows the recovery of robust tissue structures with high cohesiveness; iii) more hierarchical and stratified tissue structures were also developed using Mag-TE; and iv) the recruitment of new vessels was demonstrated using a chorioallantoic membrane assay, even in the presence of higher complexity-structures.Reproduced with permission. [90]Copyright 2013, Elsevier.Reproduced with permission. [93]Copyright 2014, Elsevier.Reproduced (adapted) with permission. [96]Copyright 2014, Elsevier.Reproduced with permission. [110]Copyright 2020, Elsevier.Reproduced with permission. [114]Copyright 2007, Wiley-VCH GmbH.Reproduced with permission. [119]opyright 2017, Elsevier.Reproduced with permission. [120]Copyright 2020, Elsevier.
cell-cell and cell-ECM contacts, the role of multicellular aggregates as providers of closer-to-physiological moieties with regenerative abilities has been widely explored.The remarkable ability of cells to self-organize in vitro into multicellular spheroids has driven not only the fabrication of regenerative units, but also the use of these structures, ranging from stem cell aggregates to specialized organoid structures, as models to study human development and disease in vitro with improved predictability, as compared to traditional cellular monolayers. [91]In general, the use of multicellular aggregates as regenerative devices has mostly focused on two possible regenerative pathways that are not necessarily independent, namely i) the use of aggregated cells as secretory vehicles of proangiogenic, trophic, and immunomodulatory molecules with high local retention efficacy, as compared to the direct injection of cell suspensions into defects (Figure 4Bi), [92] and/or (ii) the administration of healthy in vitro prepared tissue precursors or mimetics into the injured site. [93]everal methods have been used to fabricate multicellular aggregates.Starting with well-defined approaches such as hanging drops, the cells spontaneously aggregate at the bottom of droplets placed in culture plate lids turned upside-down. [94]Magnetic cellular spheroids can be engineered using the hanging drop method, in which cells are prelabeled with MNPs.The resulting spheroids can be effortlessly manipulated and separated using magnetic forces, eliminating the need for centrifugation.Kim et al. reported a straightforward and effective approach for generating spheroids using a magnetic pin array system.Magnetized cells were concentrated in a targeted direction using external magnets and magnetically induced iron pins, creating a localized magnetic field that attracted the cells.This strategy allows the reproducible generation of size-controlled spheroids.Furthermore, spheroids with versatile designs, including random mixed, core-shell, and fused spheroids, were successfully produced using this methodology (Figure 4Bii). [95]icrofluidic devices have also emerged as a new tool for the high-throughput production of multicellular spheroids with precise size control.Yoon et al. proposed a microfluidic system based on droplet technology capable of encapsulating both cells and MNPs within alginate beads to mimic the multicellular tumor spheroid functionality.Finally, the magnetic spheroids could be collected and easily transferred to the target. [96]s an alternative to traditional iron oxide MNPs, magnetiferritin nanoparticles have also been explored for the fabrication of magnetic spheroids.As a biological alternative to MNPs, these particles can avoid the need for complex surface modifications, reducing their potential adverse effects on cells. [97]he magnetism of spheroids in previous studies relied on magnetic particles previously internalized within the cells. [94,95]owever, nonconventional approaches, such as the Janus structure of magnetic cellular spheroids proposed by Mattix et al., have also been investigated.These structures had two domains, one comprising cells and the other comprising MNPs and ECM components, forming a Janus magnetic cellular spheroid (Figure 4Biii). [98]Cellular spheroids with separated cells and MNPs were successfully produced, allowing the fabrication of tissues with different cellular and extracellular compositions and contents.
One major problem in tissue constructs with high cell density is the need for the correct perfusion of essential nutrients and oxygen for tissue survival.Microvascular tissue constructs can be engineered by applying a magnetic field, which promotes an aligned vascular network.In particular, Morin et al. demonstrated the formation of the aligned sprouting of endothelial cells in magnetic spheroids, which is a crucial step in the generation of aligned microvascular networks. [99]espite the remarkable outcomes of spheroids in the engineering of tissue constructs in vitro, their applications in disease modeling and drug screening have also been widely recognized.
Owing to their superior complexity, as compared to conventional two-dimensional (2D) models, spheroid models play an important role in cancer treatment.Perez et al. demonstrated that magnetic spheroids with high sphericity can be obtained by simply promoting the magnetic aggregation of cells with internalized MNPs.Using a carcinoma cell line as a proof-of-concept, such structures have been explored in terms of cell proliferation, invasion capacity, and the influence of spheroid maturation on cell invasion and drug penetration, such as doxorubicin. [100]These tissue models were also investigated to recreate the in vivo environment of neural tissue.Using magnetic-labeled spinal cord cells, magnetic spheroids were engineered with high size and shape fidelity by only imposing an external magnetic field. [101]n all these scenarios, the fabrication of spheroids requires specific and expensive culture plates or a combination of patterned surfaces.In recent decades, magnetic levitation has emerged as an innovative TE technique.This approach involves the use of an external magnetic field to levitate and concentrate magnetized cells at the interface between air and liquid.This spatial arrangement promotes cellular aggregation and the formation of larger 3D cultures with high cellular density, the ability to synthesize ECM, and a biochemical profile comparable to that of other conventional culture systems.104] Other advanced strategies, including robot-assisted assembly, are currently used for the fabrication of uniformly sized spheroids by assembling magnetized cells.Whatley et al. demonstrated the production of 3D cell-based structures using MNPs as patterning agents to guide the attachment and self-assembly of MNP-loaded endothelial cell spheroids onto a preestablished magnetic template.

CS Engineering:
CSs have been explored as basic tissue units for engineering complex and hierarchical constructs to fabricate tissues in vitro.CS technology has emerged as a reliable alternative to conventional and limited harvesting strategies that use proteolytic enzymes, such as trypsin-chelating agents (such as EDTA), which affect cell integrity and disrupt crucial intercellular junctions and cell surface proteins. [21,105]This methodology allows the creation of cell-dense tissues with uniform cell distribution that can be harvested as a whole, preserving their ECM. [106]Upon in vivo transplantation, tissues recovered using CS engineering can adhere to the host tissue surface without suturing to establish a dense cell-like structure, [107] facilitating the preservation of essential cell-cell interactions crucial for adequate tissue regeneration. [108]Additionally, owing to its composition that resembles natural tissues, it circumvents the constraints associated with scaffold degradation, potential inflammatory responses, and limited passive diffusion (for example, inadequate delivery of oxygen and essential nutrients and insufficient removal of metabolic waste) that could hinder tissue formation. [109,110]The mechanisms of preparation, harvesting/manipulation, and transplantation of CSs were first reported by Okano in the 90s. [111,112]The mechanisms mainly involve the use of thermoresponsive culture dishes that allow reversible cell adhesion and detachment by changes in the hydrophobicity of the surface. [110]By simply lowering the culture temperature to below 32 °C for 30 min, intact CS monolayers could be collected and transplanted into the host tissue.In this context, the CS technology has been applied to various tissue types, including heart, liver, bladder, bone, cornea, and esophagus. [106]Successful clinical outcomes have been achieved, particularly in cases involving the cornea and esophagus. [113]However, these substrates face limitations such as high cost, low mechanical strength, and cellular abnormalities arising from physiological changes within the cellular microenvironment. [23]Furthermore, conventional CS engineering presents a hurdle for the creation of larger 3D tissue-like structures because of their intricate nature.The process of developing stratified and heterotypic cellular structures involves the manual stacking of previously constructed monolayers, which is time-consuming, restricts cell-cell interactions, and impairs the precise spatial positioning control of the desired cells. [108]o address this challenge, magnetic harvesting systems were first proposed by Ito et al. with the goal of producing heterotypic and stratified CSs with aided spatial manipulation. [114,115]Initially, magnetized cells were obtained by incorporating magnetic cationic liposomes with a positive surface charge, and successful particle internalization was demonstrated in distinct cell types, namely, rat hepatocytes, human aortic endothelial cells, and human retinal pigment epithelium cell lines.The magnetized cells were easily manipulated using neodymium magnets, resulting in cell accumulation toward the source of the magnet. [6,116]Ito et al. used magnetically labeled keratinocytes to demonstrate the production of stratified CSs with a 10-layer conformation.Upon removal of the magnet, the CSs were displaced from the bottom of the plates, and the sheets were harvested by placing the magnet on top of the CS. [117]This concept was explored by the same group to engineer heterotypic multilayered CSs comprising human hepatoblastoma HepG2 cells and mouse NIH3T3 fibroblasts.The results revealed major ECM components, such as FN and collagen type I, which are crucial for the development of robust tissues akin to native organisms. [108]Following the same methodology, tissue constructs with complex shapes, such as tubular structures, were produced.Briefly, when a cylindrical magnet is rolled onto a CS, it is attracted to the magnet, forming a tube around it, and a tubular structure is obtained after removing the magnet. [118]agnetic-layered CSs composed of MSCs were successfully fabricated.The ability of these tissues to differentiate into specific lineages, namely osteogenic, adipogenic, and chondrogenic, was investigated after 21 days of culture in each induction medium (Figure 4Ci). [119]When MCSs sheets were transplanted into bone defects in nude rats, new bone enclosed by osteoblast-like cells developed in the defect area.
Another main constraint in TE is the establishment of blood vessels within the implanted tissues.Angiogenic CSs were engineered using a similar approach, in which vascular endothelial growth factor (VEGF) was labeled with the magnetic cationic liposomes developed by Ito et al.After implantation of the tissue grafts in nude mice, the authors visualized the vascularization of the tissue construct, which was a thick structure with high cell density. [120]Notably, even in the absence of VEGF, MSCs sheets exhibited higher angiogenesis, as compared to the injection of isolated MSCs. [121]The introduction of arginyl-glycylaspartic acid domains into magnetic cationic liposomes facilitated cell growth, CS construction, and CS harvesting using magnetic force alone. [122]n a recent proof-of-concept study, Gil et al. demonstrated the generation of magnetic CSs using rod-and sphere-shaped amino-functionalized iron oxide nanoparticles. [123]Functionalization of nanoparticle surfaces could be useful for TE applications, including the immobilization of cell differentiation agents to control stem cell fate and antibodies for attaching biomaterials to other cells or specific proteins.This technology has been applied to monocultured CSs such as keratinocytes, cardiomyocytes, hepatocytes, endothelial cells, MSCs, and retinal pigment cells. [28]Mag-TE has also been used for the fabrication of intricate tissues that are challenging to achieve using conventional cell culture or coculture techniques.These include 2D and 3D cell layers, tubular structures, and 3D-organized assemblies comprising multiple cell types. [6,114]Gonçalves et al. reported the successful fabrication of magnetically responsive tenogenic patches based on Mag-TE using commercially available chitosan-coated MNPs and a subpopulation of tenomodulin-positive human adipose derived stem cells (hASCs) (Figure 4Cii). [124]Lu et al. developed magnetic CSs using nanoscale graphene oxide-coated MNPs.The particles were easily taken up by cells such as dental pulp stem cells, the preosteoblast cell line (MC3T3-E1), bone marrow-derived MSCs, chondrocytes, and HUVECs.The authors also demonstrated the ability to create multilayered CSs with precise control of the CS shape and structure.
However, the development of complex-shaped 3D-like constructs using living materials is hindered by the challenge of reproducing the mechanical properties of native tissues.Using MC3T3-E1 cells, magnetic CSs with diverse shapes and improved mechanical properties were successfully produced at the macroscale using a universal approach that relied on the substrate design, cell density, and magnetic force (Figure 4Ciii). [125]he resulting magnetic CSs demonstrated a Young's modulus comparable to that of soft tissues.
More recently, Mag-TE yielded impressive results in producing hierarchical 3D cohesive bone-like vascularized tissue in a simple, cost-effective, and time-saving manner, facilitating the construction and manipulation of tissues aided by a magnetic force.Notably, such 3D constructs have proven to be effective in promoting the osteogenic differentiation of hASCs in vitro while also triggering the recruitment of blood vessels with potent preangiogenic capabilities (Figure 4Civ). [115]he fate and distribution of transplanted CSs upon implantation are crucial for evaluating the regeneration process.The presence of MNPs in Mag-TE not only allows for the engineering of complex hierarchal tissues but also provides a noninvasive method to track the implanted tissue over time.Zhou et al. demonstrated that MNPs were feasible for the in vivo labeling of CSs with a tracking time of up to 12 weeks using MRI scanning. [126]Another group corroborated these findings, showing that the healing capacity of CS-derived bone marrow-derived MSCs in a digestive fistula model (mice) could be assessed using MRI.This imaging tool was used to assess the integration of CS into the host tissue, inflammation, healing status, and vascularization. [22]he regenerative potential of magnetic CSs has been investigated in tissues such as skeletal and cardiac muscles, [118,127] bone, and even angiogenesis, [120,128,129] highlighting the superior capability of this technology, as compared to artificial synthetic scaffolding materials, across a broad spectrum of tissue types.

Cell Behavior Under Magnetic Stimulation
The role of external stimuli, namely mechanical stimulation, on cellular behavior has been widely investigated in recent years.Mechanical stresses were used in a set of physiological processes, such as cell and tissue growth, tissue matrix production, and stem cell differentiation.Mechanotransduction is a well-established phenomenon in which cells transduce or convert physical stimuli into biochemical activities.MNPs have been used as nanoinstructive tools for diagnosis and therapeutics.They have been incorporated into cells or biomaterials to control cellular behavior.These nanomagnetic systems serve as mechanostimulation platforms, enabling the precise application of controlled forces to individual cells and multicellular biological tissues, thereby accurately regulating cellular functions. [130,131] magnetic field generates mechanical forces on the cell membrane that are converted into mechanotransduction signals inside the cell.The tensile strength is then delivered to the actin cytoskeleton on the cell membrane, promoting integrin-ligand binding and maturation of focal adhesions.These processes lead to the activation of several mechanosensitive and signaling proteins, which can stimulate cell adhesion, proliferation, and/or differentiation (Figure 5A).Briefly, cells can be guided toward specific biological events by regulating the mechanical forces produced by a magnetic field. [132]his concept was demonstrated using osteoblasts with MNPs adhering to the cell surface.Upon magnetic stimulation, the magnetized cells enhanced the regeneration of the bone matrix.This concept was extended to scaffold-based approaches, whereby supermagnetic scaffolds supplied the necessary cues for stimulating cell behavior by applying an external magnetic field. [133,134]For example, hydroxyapatite scaffolds encapsulating preosteoblast cells showed increased cell adhesion, proliferation, and differentiation after the incorporation of MNPs. [135]However, the mechanism governing cell behavior is still not fully understood.
Rampino et al. investigated the influence of a time-dependent magnetic field on the biological outcomes of periodontal ligament stem cells. [136]The mitogen-activated protein kinases (MAPK) cascade (p-ERK1/2) was activated under magnetic stimulation, promoting cell proliferation.The prominent effects on cell differentiation were substantiated using the metabolic profile of the cells, where the magnetic stimulus induced osteogenesis in a time-dependent manner. [136]Considering the bone physiology, this tissue requires dynamic mechanical stimulation to maintain its functionality.However, most therapeutic approaches fail to address this topic, focusing only on the biochemical and structural aspects and neglecting the pivotal role of shear stress during bone formation.Therefore, the application of dynamic magnetic environments is a feasible solution.Henstock et al. combined magnetic-labeled human MSCs (positive for TREK1 mechanosensitive ion channel) with BMP-2, and then the cells were seeded into a collagen hydrogel scaffold and delivered into the femur of a chick. [82]Using a vertically oscillating external field, the nanoparticle-receptor complex was stimulated, promoting calcium deposition and consequently biomineralizing the tissue construct.
The positive effect of the magnetic field has been demonstrated in cell-based tissue constructs, where the presence of MNPs stimulated specific biochemical responses involving ECM deposition and cell differentiation.Kikukar et al. also reported that a magnetic field could influence the morphology of magnetized cells, leading to changes in cell signaling and gene expression.The authors used MSCs to verify that the expression of osteogenic factors, such as RUNX2 and BMP-2, improved by increasing the magnetic field.The authors corroborated the presence of more adhesion domains and an elongated shape in the cell morphology (Figure 5B). [137]he influence of a PEMF on cell behavior was recently investigated.Several studies have demonstrated that PEMF stimulates specific biochemical responses. [138,139]For example, PEMF exerts a significant effect on the inflammatory process, influencing the release and expression of cytokines, thereby creating synergistic actions to guide the healing process.Such behavior was observed in tendon TE, where MNPs and PEMF prompted typical cell alignment in the tendon within cell-based constructs.Conversely, a disordered architecture may contribute to degenerative conditions facilitated by proinflammatory microenvironments (Figure 5C). [140]inhas et al. also demonstrated that PEMF has a strong modulatory effect on the inflammatory process via MAPK (ERK 1/2).The authors demonstrated the role of the PEMF in modulating the pathophysiological environment of the tendon, which is a critical step in clinical therapies for tendon regeneration. [141]EMF has been used for the proliferation and differentiation of bone marrow stem cells, specifically for facilitating osteogenic differentiation, through multiple mechanisms associated with Ca 2+ regulation.The primary effect of PEMF is closely associated with the early augmentation of the intracellular calcium concentration, which is a reliable indicator of the initial stages of osteogenic development. [142] magnetic field plays a positive role in directing the cardiomyogenesis of embryonic stem cells for the regeneration of cardiac tissue.By using an on/off cyclic magnetic force, Mary et al. demonstrated the development of the spontaneous beating of magnetized cells, which was attested by the overexpression of -actin and troponin proteins and by the upregulation of genes of the cardiac lineage (Tnnt2, Myh6, and Myl-2). [143]ulsed magnetic stimulation has also exhibited important outcomes in neural TE by supporting neural stem cell proliferation.In vivo assays showed that increasing the number of pulses per day significantly increased the proliferation of neural stem cells by modulating cell cycle progression.The therapeutic effects of repetitive magnetic stimulation have been used in the treatment of depression by avoiding a decreased number of neural cells and enhancing neurogenesis.Neurogenesis is a potential therapeutic strategy for central nerve system diseases. [144]igure 5.The role of magnetic fields on cell behavior.A) Illustrative scenario of the mechanotransduction phenomenon after the mechanical stimulation of cells.B) In the presence of the magnetic stimuli, MSCs showed an increased gene expression of BMP-2 and RUNX2.Moreover, the cell morphology also changed with more adhesion domains and an elongated shape.C) The presence of a pulsed magnetic field (PEMF) also exerted a positive effect on the typical cell alignment in a tendon within the cell-based constructs.Moreover, PEMF also influenced the expression of cytokines, including proinflammatory factors that could guide the healing process.D) The magnetic field was also applied in cancer treatment.The breakage of lysosomes via magnetic-mechanical destruction induced cancer cell death.Reproduced with permission. [126]Copyright 2018, Wiley-VCH GmbH.Reproduced with permission. [131]Copyright 2013, American Chemical Society.Reproduced under the term of CC-BY license. [134]Copyright 2019, The Authors, published by MDPI.
Magnetic stimulation has been explored for differentiating induced pluripotent stem cells (iPSCs) into neurons for the treatment of neural lesions.Through a comparative analysis of three magnetic stimuli, namely high-frequency, low-frequency, and intermittent theta-burst stimulation, the authors observed that both low-frequency and intermittent theta-burst stimulation enhanced the generation of mature neurons from human iPSCs.However, the mechanism of cell response to different magnetic stimuli has not been completely clarified and could be a limitation to the therapeutic effect of neural stem cells. [145]echanotransduction strategies have also been reported for cancer treatment because the oscillation of MNPs can induce the rupture of cancer cell membranes.As shown in Figure 5D, epidermal growth factor (EGF) peptide-modified MNPs selectively accumulate in the lysosomes of cancer cells via endocytosis. [132]nder a rotating magnetic field, the aggregated MNPs exert a mechanical force on the lysosomes, leading to localized mechanical damage to the lysosomal membrane and, consequently, cell death.
Generally, MNPs provide a unique approach for direct mechanotransduction in cells and can be easily translated into clinical practice.Materials implanted in many tissue defects tend to fail rapidly because the cells do not receive the stimulatory loading required for the integration and development of the tissue matrix. [146]MNPs can be rigorously directed toward mechanotransduction pathways by tailoring the size, coating, or even the magnetism of the particle.Therefore, the mechanical properties of the biomaterial, which is a common weakness during implantation, do not constrain the biological mechanism of mechanotransduction.
This approach has numerous advantages, as it provides a drugfree means of controlling differentiation and can be easily incorporated into various cell culturing methodologies for cell therapy.

Assembly of Building Blocks into Complex Tissues/Organoids
Regardless of the relevance and recognized potential of cellular microaggregates as single regenerative units, recent trends have highlighted their role as interesting components for integrating larger-scale tissues prepared in vitro.These mechanisms may be crucial for promoting effective regeneration of large defects.Extra structural cohesion must be produced by cells through the secreted ECM. [147]Based on this knowledge, large-scale constructs can be engineered by combining preassembled clusters such as scaffolds, CSs, spheroids, and multicellular aggregates.Cellular cross-talk occurs naturally and is essential to engineer heterogeneous cellular tissues in which intercellular communication is crucial.Three main strategies are currently used to assemble these units into complex structures, namely self-, guided, and direct assembly. [148]Considering the inherent magnetic force of the tissue building blocks fabricated using Mag-TE, large tissue constructs and/or tissues with increased complexity can be assembled using a magnetic-guided assembly strategy.Magnetism, as compared to conventional methodologies, also facilitates in vitro tissue manipulation, enabling the recovery or change of media with minimal tissue disruption. [149]he expedited assembly of shape-controlled magnetic microtissues can be accomplished using external magnetic forces, leading to the formation of macroscale tissue constructs through either random or directed assembly.
For example, Liu et al. designed circle-and clover-shaped magnetic microcryogels inspired by the "lock-and-key" design to direct microtissue assembly.The tissue constructs quickly agglomerated, forming a connection between neighboring microtissues after 5 days (Figure 2B). [83]On day 10, the individual microtissue building blocks effectively merged, resulting in an inseparable assembly.Conversely, in the absence of a magnet, scattered magnetic microtissue building blocks with minimal connections were observed on day 10, even when the microtissue building blocks were initially seeded at a high density to ensure proximity.
Ho et al. also showed that by using spheroids as tissue units, large tissue constructs can be formed by the fusion of patterned spheroids in the presence of a magnetic field. [94]To enable the fabrication of more advanced and sophisticated tissues in vitro, Whatley et al. used MNPs to guide the attachment of magnetic spheroids onto a preconstructed magnetic template, enabling the formation of 3D cell-based structures.This magneticdirected technique enabled the rapid patterning of cell spheroids based on the desired magnetic patterns, highlighting its potential for fabricating intricate and scalable 3D multicellular tissue structures. [112]Complex geometries were also produced, including tissue rings, in which a single homogenous tissue was formed without the presence of individual cellular spheroids.As stated in Section 4.3.1, this methodology allows the highthroughput fabrication of small tissue units.Complex tissue constructs have been engineered by combining cell patterning with scaffold approaches and CS engineering.Free-standing tissue building blocks were also designed with distinct geometries and sizes using a microarray composed of a superhydrophobic surface with wettable domains, mimicking the functional units of natural structures (Figure 2E). [57,58]his concept has already shown remarkable results in the fabrication of magnetic hydrogels and CSs in accordance with the methodologies described in the previous sections.Considering the strong deposition of the ECM in CSs, such microfabrication platforms allow the fabrication of robust tissues with a conserved architecture, which is a common issue in scaffold-free approaches. [58]The use of cells as building units coupled with a microfabrication platform offers the potential to fabricate intricate multiscale and multifunctional tissues with clinical significance, including therapies or disease models.
Thermoresponsive surfaces with nanotopography for providing contact guidance cues for cellular alignment were also applied in the fabrication of CSs by combing such surfaces with cells encapsulating MNPs to facilitate the magnetic levitation of CSs. [150]Fabrication of these microphysiological systems, frequently referred to as organs-on-chip or in vitro organ constructs, is mostly achieved using manual processes with high user dependency, making their reproducibility more challenging. [151]Therefore, automated processes are required in microphysiological model technologies for general industrial adoption.Bioprinting has emerged as a promising alternative to manual procedures, allowing for more controlled positioning of tissue constructs.The magnetic characteristics of magnetically labeled spheroids allow their motion over short distances with the aid of a magnetic force.By combining external magnetic spheroids with bioprinting technology, complex aggregated tissue constructs can be engineered by positioning and assembling multiple spheroids as bioinks. [101]his concept was demonstrated in neural TE, where magnetic forces enabled precise control of the spheroid position in a hydrogel template.This approach has enabled the development of neural constructs that replicate the hierarchical organization of the central nervous system, with cell populations that project axons toward distant populations.Bioprinting has also been explored for the magnetic design of several stem cell tissues.Encouraging results have been observed in the context of engineering vessels de novo and in guiding stem cells toward the chondrogenic pathway to produce cartilaginous tissue.The application of magnetically induced cyclic stretching and compression on stem cell based tissues has shown potential in inducing cardiomyogenesis, offering a facile and efficient approach for cardiac tissue regeneration. [152]able 1.Regenerative medicine clinical trials involving the application of magnetic fields.

Clinical trial Condition Treatment Phase Status
Dermal profile analysis using NMR-MOUSE Hypotension during dialysis disturbance; balance; fluid Dermal NMR-profile measurement -Terminated [ 159] Predictive factors of pelvic NMR in the response of arterial embolization of uterine leiomyoma Uterine leiomyoma MRI -Completed [ 160] Short-and long-term exposure to unique, time-varying pulsed electromagnetic fields in refractory carpal tunnel syndrome Refractory carpal tunnel syndrome neuropathic pain neuromodulation Reduction of pain scores using magnetic energy Phase 4 Completed [ 161] Evaluation of the effect of biophysical stimulation with pulsed electromagnetic fields on intraspongious bone edema in anterior cruciate ligament reconstruction Anterior cruciate ligament injury pain Specific pulsed electromagnetic fields -Recruiting [ 162] Assessing impacts of static magnetic fields on peripheral pulses and skin blood flow Peripheral arterial disease; arterial stiffness Magnet and sham -Recruiting [ 163] Therapy of toxic optic neuropathy using a combination of stem cells with electromagnetic stimulation Toxic optic neuropathies Stem cells with electromagnetic stimulation Phase 3 Completed [ 164]

Clinical Trials
Cell-based therapies mostly relying on the direct injection of single-cell suspensions have been clinically applied in recent decades.However, TE is currently considered an excellent strategy for regenerative therapies, with 63 ongoing clinical trials focusing on collagen matrices and CSs. [153]Only six clinical trials in regenerative medicine involving the use of magnetic fields are ongoing.Safety concerns regarding the use of MNPs and a magnetic field on TE have hampered their translation into clinical practice.The mechanism underlying MNP degradation in vivo is still poorly explored, leading to the apprehensive use of MNPs in biomedicine.Therefore, the toxicity profiles of MNPs and their degradation over time must be extensively investigated to clarify the benefits and adverse effects of Mag-TE.Changing several features of MNPs, such as their shape, size, structure, and surface, enable the identification of the most suitable MNP design for specific applications.Several studies have suggested that surface modifications can significantly improve the biosafety of MNPs. [154]Previous research has indicated that despite physical modifications and atomic-scale degradation of MNPs over time, no observable toxic effects have been detected in living organisms.Numerous reports have suggested that MNPs exhibit biocompatibility because of their ability to integrate into endogenous iron-related metabolic pathways.Furthermore, the ionic species released during the degradation of MNPs can be used by cells for the biosynthesis of new MNPs, known as endogenous MNPs. [155]This phenomenon can be attributed to the inherent presence of magnetic particles in human organs, including the brain, heart, spleen, liver, ethmoid bone, cervical skin, and tumors. [156]o address potential concerns related to long-term procedures, one possible solution involves the implementation of a protective shell to prevent the degradation of the magnetic core. [157]dditionally, novel bioinspired synthesis methodologies hold promise for addressing long-term concerns and offer alternative approaches to enhance the durability and stability of magnetic components in various applications. [156]egarding the risks associated with the aggregation of MNPs in the bloodstream, Wilhelm et al. demonstrated that cells can effectively regulate the redistribution of nanoparticles.Specifically, they observed a timely transition of nanoparticles from a densely assembled state within early endosomes to a more dispersed and exposed state within lysosomal compartments. [158]This mechanism delays the release of free cytotoxic iron ions, confining them solely to the lysosome, where ferritin proteins store the released iron in a safe and nontoxic form.However, the exact underlying mechanism remains unclear.Therefore, it is crucial to establish stringent and clearly defined guidelines to promote the effective and responsible use of magnetic approaches in the regenerative medicine field.Table 1 lists selected information on ongoing studies using magnetic fields.
The main outcomes of magnetic fields in clinical practice rely on MRI for monitoring.For example, the assessment of the fluid status in patients on dialysis is a significant clinical challenge.Normally, fluid management for these patients relies primarily on monitoring differences in body weight.However, this approach can lead to complications such as hypotension if excessive fluid is removed during dialysis or symptoms associated with fluid overload if the patient retains excessive fluid.A methodology using a mobile noninvasive NMR spectroscopy measurement (NMR-MOUSE) setup was developed to measure the hydration status of the skin, thereby monitoring the fluid levels in patients during dialysis. [159]RI is also useful for determining predictive factors for arterial embolization of uterine leiomyomas during pelvic MR.This study monitored symptomatic premenopausal women diagnosed with uterine leiomyoma who underwent uterine artery embolization (UAE).During the treatment process, MRI was conducted to assess the volume of both the entire uterus and leiomyomas one month prior to UAE and six months posttreatment. [160]he radiological dimensions of the leiomyoma and uterus as well as their respective volumes were assessed.Additionally, the number of leiomyoma fibroids and their specific locations within the myometrium were classified.PEMF has been approved by the US Food and Drug Administration for the treatment of nonunion fractures, neurogenic bladder, and musculoskeletal pain.PEMF demonstrates a positive effect on the reduction of neuropathic pain caused by carpal tunnel syndrome.Considerable evidence supports the notion that time-varying magnetic fields can generate biological effects by safely stimulating extremely low-frequency electrical currents within the tissues.The data showed that neuropathic pain was significantly reduced by directing the PEMF to the carpal tunnel region. [161]he potential role of PEMF in the reduction of postoperative pain by modulating inflammation in patients with knee-bone bruises has also been explored.This study aimed to examine the effects of PEMF on reducing the size of the bruised bone area, enhancing knee function and reducing the requirement for anti-inflammatory medication during the postoperative period, as compared to the control group. [162]tatic magnetic fields have also been considered in biomedical applications.An ongoing study aims to investigate the effects of static magnetic fields generated by a small magnet on peripheral pulses and skin-blood flow.To achieve this, a neodymium magnet and a sham device are positioned near the ulnar and medial arteries at specific sites.The impact of this placement on the peripheral pulses will be evaluated using photoplethysmography of the fingers, whereas skin blood flow will be assessed using laser Doppler perfusion measurements. [163]agnetism can be used to treat several conditions by combining stem cells with electromagnetic stimulation.Sildenafil, a vasoactive medication commonly used to treat erectile dysfunction, typically decreases blood flow to the optic nerve head and contributes to neuroinflammation, potentially leading to permanent vision impairment.In contrast, Wharton's jelly-derived MSCs (WJ-MSC) enhance mitochondrial adenosine triphosphate synthesis through paracrine effects and suppress neuroinflammation through their immunomodulatory properties.Repetitive electromagnetic stimulation (rEMS) has the potential to modulate the ion channel equilibrium and promote axoplasmic flow reorganization.The combination of WJ-MSC and rEMS was explored for the treatment of toxic optic neuropathies. [164]

Conclusions
Advances in TE over the last few decades have enabled the development of tissues with structural and compositional accuracy.However, achieving functional reproduction of native tissues remains a significant hurdle in the clinical translation of TE.Recent advancements in this field include the generation of new "smart" materials due to their precise and selective response under external signals or the surrounding environment.Among them, tremendous progress has been achieved using MNPs, including those included in cells and scaffolds and/or acting as magnetic carriers of drugs and biomolecules.Therefore, functional 3D tissues can be fabricated using Mag-TE and these magnetized units as building blocks.Other competencies, such as the influence on cell behavior, can also be supported by Mag-TE, which affects the adhesion, proliferation, and differentiation of cells.Smart drug delivery systems can be developed by controlling the release of drugs and growth factors using remote magnetic fields.For in vivo implantation, tissues produced in vitro can be easily transplanted and securely positioned at the site of injury under the guidance of an external magnetic field.The scaffold-free Mag-TE strategy yielded remarkable results in the fabrication of functional tissue constructs.Because of the high initial cell density in this approach, the time required for tissue formation is dramatically reduced, and the proliferation and migration of cells are not crucial parameters.Moreover, the strong ECM deposition in these constructs ensures the development of highly co-hesive structures, enabling the creation of cell-based assemblies with complex architectures that mimic the native tissue environment.
Generally, small tissue building blocks, such as CSs, spheroids, and tissue strands, serve as the foundation for bottom-up strategies employed to create complex tissues and organoids.This process involves the controlled assembly of heterogeneous building blocks composed of various cell types.However, concerns regarding the immobilization time required for the initial fusion of these building blocks and the limitations imposed by their inferior mechanical properties hinder the broader application of this approach.Such problems are addressed using scaffold-based strategies, in which scaffolds act as cell protectors during tissue formation.Moreover, the mechanical behavior of tissues can be easily adjusted by changing the scaffold constituents.However, the superior mechanical properties of these materials can dramatically affect cell adhesion, migration, and spreading, leading to inhomogeneous structures and compromised tissue function.
Considering an ideal scenario for TE, the authors envisage the fusion of scaffold-based and scaffold-free approaches, which are usually considered as rivals, into a synergetic TE strategy.
Briefly, the integration of different technologies and materials into an operational strategy, together with the ability to modulate the properties of materials (chemically, physically, or in terms of biodegradability), will permit the development of elevated living structures through bottom-up approaches, enabling the construction of complex functional tissues with relevant physiological architectures for clinical applications.
Additionally, the authors believe that rapid clinical acceptance is strongly correlated with the ability to define a universal strategy whereby only modulating the properties of the scaffolds can be further extended to several cell and tissue types without considerable modifications to the assembly process.Accordingly, it is anticipated that such synergy will accelerate in situ tissue assembly, profiting from the development of improved living materials designed to meet patient needs.
Further association of this concept with a magnetic field could provide additional control over tissue formation with tunable tissue size, shape, and architecture, contributing to the development of functional tissues that more closely replicate the native environment, as described in the literature.Intrinsic magnetism offers advantages such as easy manipulation, transplantation, and tissue monitoring over time, prompting its application in clinical practice.Additionally, the authors anticipate that the robotic-assisted deposition of magnetic building living blocks could further increase the accurate control of their positioning in 3D space, allowing faster and more accurate representations of natural tissues within a denser tissue microenvironment, inherent ECM secretion, and a prevascularized network, as evidenced in engineered magnetic tissues.This new concept exhibits excellent potential as an optimal solution for addressing the broad spectrum of challenges currently faced in TE, providing several opportunities for overcoming individual bottlenecks and developing new solutions that leverage the added value it offers.However, further translational studies are necessary to validate the efficacy and advantages of MNPs, thereby enhancing their potential use in TE and regenerative medicine.Therefore, to further advance this idea, it is essential for biologists, materials scientists, www.advhealthmat.deengineers, product designers, and medical doctors to cooperate to push the boundaries of what can be accomplished in the field and to help develop relevant clinical solutions.

Figure 2 .
Figure 2. Cell patterning strategies to guide tissue formation.A)The patterning of single cells was achieved using a magnetized pin holder device, promoting the formation of cord-like structures, which are crucial during angiogenesis.B) Similar devices were used as a cancer model to investigate the invasive capacity of BALB/3T3 cells.C) Three-dimensional (3D) tissue constructs were also fabricated using this strategy, where magnetic spheroids formed à priori were guided to a prefabricated magnetic template.Finally, the spheroids fused and a 3D tissue with the geometry of the template was formed.Reproduced (adapted) with permission.[50]Copyright 2009, Wiley-VCH GmbH.Reproduced with permission.[52]Copyright 2009, Royal Society of Chemistry.Reproduced with permission.[53]Copyright 2014, Wiley-VCH GmbH.

Figure 3 .
Figure 3. Fabrication of magnetic tissues using the scaffold-based approach.A) Schematic illustration of the scaffold-based strategy, where magnetized cells are seeded or encapsulated in scaffolds composed of natural, synthetic, or hybrid materials.B) General view of hydrogel composites-Fe 2 O 3 /hyaluronic acid (HA)/polyvinyl alcohol (PVA) (top).SEM images of porous hydrogels, where bone mesenchymal stem cells (BMSCs) adhered and aggregated in the pores.A small number of cells adhered to the porous surface of the control (bottom).C) Micrographs of a hybrid hydrogel containing collagen type II, HA, polyethylene glycol (PEG), and MNPs (top).These materials were explored for cartilage TE, where the collagen was extracted from sheep femur condyle with potential calcification (bottom).D) Micropatterned platform for the high throughput fabrication of microtissues with complex geometry (on left); these magnetic hydrogels can be used to produce larger structures using magnetic-guided assembly (right).E-i) Engineering of macro-scale tissues by random or shape-directed assembly of magnetic microcryogels under magnetic force, and ii) photographs of circle and clover magnetic microcryogels and their accurate assembly.Even at the macroscale, the tissue constructs remained viable (live/dead images in Figure4Biii).Reproduced with permission.[55]Copyright 2016, Wiley-VCH GmbH.Reproduced with permission.[76]Copyright 2018, American Chemical Society.Reproduced with permission.[77]Copyright 2015, American Chemical Society.Reproduced with permission.[81]Copyright 2014, Royal Society of Chemistry.

Figure 4
Figure 4. Fabrication of cell-based tissue constructs using scaffold-free approaches.A) Image illustrating the scaffold-free TE strategy, where magnetized cells are used as building blocks for the fabrication of cell-based tissue constructs, namely, spheroids, CSs, and multishaped tissues.B) Production of magnetic spheroids using Mag-TE.i) By using magnetic spheroids as tissue units in the presence of a magnetic pin, the fusion of spheroids into a single tissue construct was accelerated and the union of the spheroids was achieved after 24 h; ii) sequential tissue fusion of vascular tissue spheroids embedded in collagen type I hydrogel, and iii) magnetiferritin nanoparticles in magnetic cellular spheroids.Magnetic-labeled spheroids were assembled onto a ring magnet.The presence of a strong magnet was crucial for the spheroid fusion in the desired shape.The inner diameter contraction was visualized in the presence of a weak magnet.C) Fabrication of CSs using Mag-TE.i) Transplantation of an MSC sheet (single layer) to the cranial bone of nude rats using an electromagnet (on bottom); ii) simple magnetic harvesting process that allows the recovery of robust tissue structures with high cohesiveness; iii) more hierarchical and stratified tissue structures were also developed using Mag-TE; and iv) the recruitment of new vessels was demonstrated using a chorioallantoic membrane assay, even in the presence of higher complexity-structures.Reproduced with permission.[90]Copyright 2013, Elsevier.Reproduced with permission.[93]Copyright 2014, Elsevier.Reproduced (adapted) with permission.[96]Copyright 2014, Elsevier.Reproduced with permission.[110]Copyright 2020, Elsevier.Reproduced with permission.[114]Copyright 2007, Wiley-VCH GmbH.Reproduced with permission.[119]Copyright 2017, Elsevier.Reproduced with permission.[120]Copyright 2020, Elsevier.