(J-381) Elucidating the effects of magnetic fields on muscle regeneration using 3D bioengineered muscle tissues

Introduction:: As a non-invasive intervention, magnetic field therapy holds great promise for promoting the tissue healing of tissues, including skeletal muscles. Although previous studies have suggested that magnetic field stimulation modulates cell behavior such as myoblast differentiation, a better understanding of whether and how magnetic fields affect muscle regeneration is much needed. This study investigated the effects of magnetic field stimulation on muscle stem cell behavior and muscle regeneration. We hypothesized that ultralow-intensity magnetic fields enhance muscle regeneration by promoting muscle progenitor cell (MPC) migration and amplification. To test our hypothesis, we first investigated the effects of magnetic field strength on MPC migration using scratch wound healing assays. We then established a three-dimensional (3D) muscle regeneration model to evaluate whether magnetic field stimulation could enhance muscle regeneration.


Materials and Methods:: We developed a magnetic field stimulation chamber to generate uniform magnetic fields within a multi-well plate area in the center (Fig. 1A). Magnetic field stimulation was controlled using a Raspberry Pi-modulated Helmholtz coil (x, y, z). The field strength was measured using a Hall sensor that provided continuous feedback to the Raspberry Pi to maintain the set field. Control groups were the samples under the influence of Earth's magnetic field. For wound healing assays, we focused on two outcome measures to assess cell migration: 1) distance in scratch and 2) F-actin expression. A shorter scratch distance indicates faster wound healing and cell migration, whereby F-actin expression negatively correlates with cell migration. Next, 3D biomimetic muscle tissues were prepared using MPCs isolated from 3–5-month-old male mice. MPCs were embedded into hydrogel mixtures of fibrin and Matrigel and pipetted into a polydimethylsiloxane chamber containing two fixed anchors. We previously demonstrated that this setup mechanically guides the formation of aligned, contractile myotubes. The 3D tissues were cultured in myogenic differentiation media for two weeks to induce myotube formation and tissue maturation. To generate a muscle regeneration model, 3D muscle tissues were injured by cardiotoxin (0.4 μM for 5 hours), causing myotube fragmentation and a reduction in contractile forces. We have demonstrated that the 3D muscle tissues displayed capabilities to regenerate after cardiotoxin injury, offering a high throughput way to assess the effects of magnetic field on muscle regeneration.


Results, Conclusions, and Discussions:: After the wound was made, cells were exposed to static magnetic fields for 24 hours with the magnetic field strength varying from 10 μT to 1400 μT. Our results showed that scratch distance and F-actin expression significantly decreased in the 400, 800, and 1000 μT stimulation groups compared to the control group. We then chose 400 μT to investigate whether magnetic field affected muscle regeneration. The injured muscle constructs were exposed to a 400 μT magnetic field applied in a direction parallel to the aligned myofibers (Fig. 1B). As muscle stem cell expansion is one of the critical initial steps of muscle regeneration, we quantified the percentage of quiescent (Pax7+/MyoD-), proliferating (Pax7+/MyoD+), and differentiating (Pax7-/MyoD+) muscle stem cells in the injured muscle constructs after one day of magnetic stimulation (Fig. 1C). As shown in Fig. 1D, exposure to magnetic fields resulted in an increase in proliferating cells (p=0.052) and differentiating cells (p=0.021). We then quantified the functional recovery of injured muscle constructs after seven days of magnetic stimulation. The contractile function (tetanic forces and force-frequency relations) of muscle constructs was significantly higher after magnetic stimulation when compared to the non-stimulated control (Fig. 1E, F). Together with 2D results, our data suggest that magnetic stimulation (400 μT) enhanced muscle regeneration through accelerated MPC migration and amplification. Overall, our study demonstrates that magnetic field stimulation can be considered as an intervention for promoting muscle repair.


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