Contactless Magnetically Responsive Injectable Hydrogel to Guide Aligned Tissue Regeneration

<b>INTRODUCTION</b><br/>Human tissues have well-defined architectures and mechanical properties that play a crucial role in their functionality. Specifically, cellular alignment is fundamental in several tissues and organs such as spinal cord, muscle, tendon, and cartilage. Several well-oriented scaffolds, able to reproduce the crucial tissue microarchitecture and to sustain regenerative process, have been produced by various tecniques (e.g. bioprinting, electospinning, freeze-drying), but very few have shown translational applications also due to the invasive surgery necessary to be implanted. Injectable hydrogels can be transplanted <i>via</i> minimally invasive injection, but usually without displaying any aligned structure. In the present work, we developed an injectable hydrogel with anisotropic architecture by applying a static magnetic field (SMF), based on gellan gum (GG), a biocompatible and biodegradable polysaccharide, hyaluronic acid (HA) and magnetic collagen fibers (MagC) for skeletal muscle regeneration.<br/><b>METHODOLOGY</b><br/>GG (1%) hydrosol added of sodium citrate (0.1%) and HA (0-0.6%) can be injected at 37°C. The gelation occurs in the presence of cations (e.g. PBS, cell culture media, biological fluids) in few minutes. MagC are prepared by mixing the 1mg/mL magnetic nanoparticles to collagen type I at pH 7.4 in different ratios (1:1, 1:3), then they are added to the hydrosol. Mechanical properties were investigated by rheometer and dynamic mechanical analysis. An extensive 3D in vitro cell culture was performed to study cell muscle behavior (viability, differentiation, elongation) and immunomodulation of human and murine macrophage response by ELISA and flow cytometry analysis. Then the hydrogels were subcutaneously implanted and aligned in Wistar-Han rats. At 1, 3 and 7 days, the hydrogels and the surrounding tissue were processed for histological and immunohistochemical analyses. Potential toxicity was evaluated by analysing the spleen, liver, kidney, and lymph nodes. <br/><b>RESULTS</b><br/>The hydrogel could be easily extruded by a 30G needle. MagC embedded into the hydrogels were easily aligned by a short (&lt;10 min) application of an external SMF (400mT, distance of ≈4cm) and, once gelated, it showed mechanical properties comparable to muscle tissue (12-19 kPa), and stress relaxation behavior was similar to human tissues when 0.3% HA was added. Additionally, the hydrogel did not negatively affected muscle cells viability and morphology, it enhanced their elongation on the magnetic fibers, and modulated macrophage polarization. Even in vivo, the hydrogel was easily injected and aligned, and no local or systemic immune reaction was detected.<br/><b>CONCLUSION</b><br/>In the present work, we developed a contactless magnetically responsive injectable hydrogel to direct aligned musculoskeletal tissue regeneration.<i> In vitro</i> analysis pointed out the system biocompatibility that was confirmed during the <i>in vivo</i> test, in which also was highlighted its ability to achieve an aligned architecture by a remote SMF application during the gelation. <br/>The versatility of the proposed system can be expected to find application to several different minimally-invasive or endoscopic surgical interventions on different body parts that might benefit from the injection of anisotropic and bioactive materials, such as the heart, cartilage, brain, spinal cord.<br/><b>REFERENCES</b><br/>- Rose J.C., Adv. Healthc. Mater., 2018;7(6):e1701067).<br/>- Das M., J. of Drug Deliv. Sci. and Tech., 2020;56,A101586)<br/>- Discher D.E., Science. 2009;324(5935):1673-7<br/>- Chaudhuri O., Nature Mater. 2016;15,326–334