A cellular response to environmental stimuli sensed at a focal adhesion level involves both mechanical changes and the participation of signaling cascades. The Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-mediated signaling cascades play an important role in mechanotransduction, with implications for the development, organ growth, and lineage specification of MSCs [83,84]. YAP and TAZ are transcriptional regulators that act as effectors for multiple cascades. On stiffer matrices, focal adhesions are more spread out and cells establish more contacts with the substrate, thus creating tension at the cytoskeleton level and creating spatial rearrangements. These events, along with the biochemical modulators, further determine the activation and translocation of the YAP/TAZ factors from the cytoplasm to the nucleus, where they stimulate gene transcription. Otherwise, if cells sense a soft ECM or substrate, mechanical tension is decreased, and YAP/TAZ are relocated to the cytoplasm, where they suffer proteasomal degradation. FAK is a functional component in focal adhesions that is activated when cells sense mechanical stress and forms complexes with members of the Src kinase family. These complexes further trigger a downstream signaling cascade involving multiple phosphorylations, which can regulate actin polymerization and PI3K and ERK pathways, leading to YAP translocation [83,85]. Considering the fact that interactions with magnetic materials and magnetic fields induce mechanotransduction regulation mainly though focal adhesions, YAP/TAZ transcriptional control was reported to be correlated with the biological response of cells [35,54,86,87]. Stem cells cultivated on magnetic scaffolds or exposed to a static magnetic field displayed an increase in YAP/TAZ nuclear localization, which was influenced by the state of actin polymerization and the spatial distribution of the cytoskeleton network [35,86]. YAP/TAZ activation after magnetic stimulation has positive effects on osteogenic differentiation and mineral deposition, but depolymerization of the actin filaments determines cytoplasmic shuttling, highlighting the essential role of the cytoskeleton in cell regulation [86]. Another proposed mechanism during osteogenic differentiation involves the activation of the mitogen-activated protein kinase (MAPK) pathway in response to the mechanical stress induced by MNPs and a magnetic force. Therefore, YAP and RUNX2 transcription factors are translocated to the nucleus, where they upregulate the expression of the collagen type I, alpha 1 chain (COL1A1), OPN, and bone gamma-carboxyglutamate protein (BGLAP) and downregulate the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARG) [54]. Moreover, MAPK-mediated pathways were reported to be activated in response to interactions with magnetic substrates without being correlated with YAP/TAZ transcriptional control. MSCs cultivated on 3D magnetic nanocomposites showed enhanced adherence and an upregulated expression of the integrins FAK and ERK1/2, indicating the activation of this signaling cascade which further promoted the transcription of alkaline phosphatase, OPN, BMP-2, and other osteogenic markers [33]. A static magnetic field was proved to have stimulatory effects toward the proliferation of stem cells, which is mediated by the activation of p38/MAPK and which regulates cytoskeleton reorganization [34]. Furthermore, in mice, an RMS treatment induced the activation of the PI3K/Akt pathway with the subsequent upregulation of glutamate trasporter-1 (GLT-1), allowing for the clearance of glutamate. Thus, a reduction in oxidative stress and neuronal damage was observed, along with improved cognitive function [88]. However, the moderate production of reactive oxygen species (ROS) seems to positively regulate the neuronal differentiation of MSCs induced by electromagnetic field stimulation. The PI3k/Akt pathway is activated in response to EGFR phosphorylation, an event that further determines the phosphorylation of the cyclic AMP response element-binding protein (CREB). The ROS scavenger decreased EGFR activation and inhibited downstream effects, suppressing the neuronal differentiation of MSCs [29].

Magnetic cues have been shown to have a great influence on stem cells’ differentiation potential, with increased susceptibility toward the specifications of certain lineages. For example, in the case of ASCs, Maredziak et al. (2016) reported that a static magnetic field increased osteogenic differentiation and the expression of specific markers, but diminished their ability to undergo adipogenesis [41]. Indeed, many studies have approached magnetic stimulation by concentrating on osteogenic differentiation. The beneficial effects observed in this type of specification might be attributed to the mechanical characteristics of the bone, which is a hard tissue. Rigid matrices with reduced elasticity were proven to influence and direct the differentiation of MSCs toward an osteogenic lineage [20]. Looking into MSCs, the mechanosensing machinery of the cells can dictate their differentiation toward osteoblasts or adipocytes by modulating YAP/TAZ transcriptional regulation depending on the stiffness of the substrate [83]. Furthermore, there is evidence indicating that osteogenesis is positively regulated by the overexpression of integrins, which is accompanied by changes in cytoskeleton arrangement and the activation of specific pathways [89].

Considering the previously discussed effects of magnetic stimulation on mechanotransduction, magnetic cues and materials have emerged as a valuable resource for the promotion of osteogenesis, with great potential in bone tissue engineering [33,90]. For example, MNPs were incorporated into a calcium phosphate cement by obtaining an osteoinductive scaffold that was seeded with human dental pulp stem cells [91]. Exposure of this bioconstruct to a static magnetic field significantly increased the osteogenic differentiation of stem cells, as shown by the increased expression of osteogenic markers and alkaline phosphatase activity. Further, differentiated cells had higher yields of bone mineral synthesis compared to those seeded on calcium phosphate cement alone or on unmagnetized scaffolds [91]. Furthermore, in vivo studies showed promising results for both magnetic materials and magnetic fields in promoting bone regeneration. Gene therapy was used to stimulate angiogenesis and osteogenesis after a bone implant in rabbits. Magnetic microspheres and magnetic fields were used to facilitate the transfection of plasmids expressing vascular endothelial factor (VEGF) [92]. In diabetic mice, a static magnetic field prevented trabecular and cortical bone deterioration, increased the number of osteoblasts, and decreased the proportion of osteoclasts. Additionally, the magnetic field treatment led to the enhanced expression of bone-specific proteins, such as BMP and osteocalcin [90]. Possible applications for musculoskeletal regeneration were reported regarding chondrogenic [87,93], tenogenic [35], and myogenic [94] differentiation. It seems that magneto-responsive scaffolds and magnetic fields have synergistic effects that promote tenogenesis by increasing specific markers such as tenomodulin, scleraxis, and decorin [35,95]. This approach, combined with the activation of activin receptor type II (ActRIIA), allowed for the modulation of the transforming growth factor β (TGF-β)/Smad2/3 signaling pathway [95]. Other experiments were also conducted for the purpose of studying cardiac [96], vascular [97], and nervous tissue [11] approaches. 

Studies on neurogenesis emphasize its promising results for the use of magnetic fields for increasing NSC differentiation, proliferation, and maturation [65,98]. These effects might be mediated by calcium channels’ activity and calcium signaling, which is suggested by the upregulated expression of the Ca(v)1 channel and increased cyclic AMP response element-binding protein (CREB) phosphorylation. The inhibition of the Ca(v)1 channel significantly decreased the differentiation and maturation of NSCs [99]. Another calcium channel, N-methyl-D-aspartate (NMDA), was reported to be upregulated during the differentiation of neural progenitor cells, leading to the overexpression of the CREB-regulated c-fos protein [100]. Furthermore, in embryonic NSCs, electromagnetic field exposure promotes neurodifferentiation through the upregulation of transient receptor potential canonical 1 (TRPC1), which modulates Ca²⁺ uptake. The increase in intracellular calcium levels was accompanied by the upregulation of NeuroD and Neurogenin1 [101]. An electromagnetic field was reported to promote the neuronal differentiation of MSCs and NSCs, and was accompanied by phenotypic and electrophysiological changes. Transcriptome analysis of both cell types highlighted significant changes in the global gene expression profile that were mediated by the upregulation of transcription factors such as hairy and enhancer of split-1 (HES1), early growth response protein-1 (Egr1), and DNA-binding protein inhibitor ID1. Among them, Egr1 proved to be a key regulator that synergizes with the electromagnetic field exposure to promote neuronal differentiation [66]. MNPs internalization with further magnetic stimulation promoted in vitro neural differentiation of ESCs, synergizing with biochemical inducers [102], and allowed guidance of neurite outgrowth for primary leech neurons [103]. Besides neuronal differentiation, some studies indicated magnetic stimulation and MNPs as a potential approach for nerve TE and spinal cord injuries [104,105,106]. Such cues were shown to promote peripheral nerve regeneration in rats by increasing the survival of dorsal root ganglia (DRG) neurons, and improving myelination and functional recovery [104,105]. These outcomes are also mediated via stimulation of a pro-regenerative phenotype of Schwann cells, which have a critical role during repair since they secrete neurotrophic factors and are responsible for myelination [107,108].

Interactions established between cells and MNPs have created the opportunity for directed cell delivery for therapeutic purposes. This aspect is of high interest for nervous TE since it could facilitate cell transplantation at inaccessible lesioned sites. For this purpose, MSCs were investigated as promising candidates for spinal cord injuries. Labeling MSCs with superparamagnetic iron oxide nanoparticles (SPIONs) proved to have no significant cytotoxic effects and allowed for in vivo tracking using magnetic resonance imaging (MRI) [109]. Cell guiding was achieved by Tukmachev et al. (2015) who developed a noninvasive magnetic system with two magnets flanking the spinal cord over the injury site. MSCs were labeled with SPIONs coated with poly-L-lactic acid and intrathecally administered near the lesioned area. Results from a histological analysis showed that the presence of magnetic stimulations led to MSC concentration and infiltration at the lesioned site, whereas in its absence, cells were evenly distributed throughout the spinal cord [110]. For brain injuries, NSCs were labeled and used for cell therapy. Studies showed that nanocomposites containing SPIONs do not affect NSCs’ potential of differentiation toward both neurogenic and glial lineages [111], whereas magnetically guided NSCs display better viability in vivo and have a precise location at the brain level [112].

Given the low capacity of nervous tissue to regenerate, bioactive molecules emerged as efficient therapeutics to stimulate the regeneration process. Therefore, strategies for precise administration of such molecules are being investigated for the central and peripheral nervous system, but they represent a real challenge. When designing magnetic vehicles for gene and drug delivery, one must take into consideration if they target the peripheral nervous system (PNS) or central nervous system (CNS), since passing the brain–blood barrier (BBB) or blood–cerebral spinal fluid barrier requires different modifications than for PNS administration. In cases of damaged tissue, MNPs could pass CNS barriers freely, but for neurodegenerative disorders, they might need functional groups to bind cell receptors [113]. MNPs functionalized with osmotin could pass the BBB without inducing any damage or cytotoxic effects. Moreover, they could attenuate memory and tau phosphorylation in an Alzheimer model [114]. Furthermore, Niu et al. (2017) combined small RNA with growth factor delivery and obtained promising results in a Parkinson model. MNPs were functionalized with nerve growth factor (NGF) and short hairpin RNA (shRNA) against α-synuclein, decreasing the number of α-synuclein-positive neurons [115].

Scaffold-based approaches for nervous tissue regeneration were mainly utilized for the peripheral system and spinal cord injuries, as these compartments require precise cell alignment and are protected by connective tissues. MNPs are usually incorporated into polymeric scaffolds, with hydrogels being intensely used [1,113]. The concentration of MNPs is an important factor that needs optimization, as it can affect cell viability and differentiation. Collagen-based coatings enriched with 0.5% MNPs showed better results in terms of cell adherence, viability, and the neural differentiation of pluripotent stem cells as compared to higher contents of up to 4% [116]. Furthermore, xanthan-based scaffolds with moderate contents of MNPs promoted cell adhesion and proliferation. Interestingly, while neat scaffolds presented a higher percentage of differentiated neurons expressing microtubule-associated protein 2 (MAP2) and Tuj1, materials with MNP content displayed more cells that were positive for synaptophysin and increased electrical transmission. Therefore, it can be assumed that the presence of MNPs stimulates the differentiation of functional neurons [117]. Rose et al. (2017) developed a scaffold-based system, called Anisogel, based on SPIONs. Microgels were loaded with SPIONs and dispersed into a hydrogel matrix precursor, and then were aligned by applying an external magnetic field. The obtained system was further tested on fibroblasts and nerve cells. This approach allowed for the precise control over the topographical and mechanical cues necessary to direct cell growth. Compared to randomly oriented microgels, cells cultivated on the aligned microgel system displayed a better orientation of neurite outgrowth and they grew in an aligned way, emphasizing the good effects of the pre-existing topographical cues. [11]. SPION-loaded polycaprolactone (PCL) nanofibers, which were magnetically oriented to form an injectable hydrogel, could support and enhance the neural differentiation of olfactory ectomesenchymal stem cells. The anisotropic nature of the resulting hydrogels, which shares similarities with the nerve tissue architecture, in addition to the presence of magnetic structures, might mediate the beneficial effects on stem cell differentiation [8]. Consistent with these results, nanotopographical cues were reported to play an important part during the neuronal differentiation of ESCs and MSCs, with an influence on nuclear morphology and epigenetic regulation in the first 24 h of a cell–substrate interaction [62]. Another magnetic hydrogel system was developed by Tay et al. (2018), allowing for the neuromodulation of primary dorsal root ganglion neurons. It was observed that a short magnetic stimulation led to the activation of transient receptor potential vanilloid (TRPV) and piezo-type mechanosensitive ion channel component 2 (PIEZO2) channels, increasing calcium uptake [12].