Poster BB17 - Myogenic maturation of skeletal muscle progenitor cells in FibMA-fibrin double network (DN) hydrogel filaments

Introduction: A next generation of skeletal muscle cell therapies is now being explored using autologous muscle stem cells incorporated into a provisional hydrogel scaffold formed into filament structures that mimic myofibers. Although a significant number of synthetic and biological hydrogels have been developed for muscle cell therapy, not every material will have suitable biological and physical characteristics that capture the dynamics and complexity of native skeletal muscle extracellular matrix (ECM), nor are they amenable to the requirements of forming filament structures by extrusion bioprocessing. The goal of this research was to investigate muscle stem cell response to biophysical, microstructural and biological properties of an encapsulating filamentous hydrogel milieu towards obtaining rapid myogenic maturation and myotube formation. Towards this goal, an interpenetrating polymer network (IPN) hydrogel was formulated as a double network (DN) with control over biological, microstructural and viscoelastic mechanical properties. The DN hydrogel was specifically designed to support myogenesis of encapsulated muscle progenitor cells and to be amenable to filament formation with sub-millimeter scale.

Materials and

Methods: The DN hydrogel was prepared from methacrylated fibrinogen (FibMA) and fibrin. FibMA was prepared by conjugating methacrylic groups to the free amines on fibrinogen lysines. The formation of the FibMA-fibrin DN hydrogels was done by a tandem crosslinking reaction first by thrombin activation of unmodified fibrinogen to fibrin, and then by a radical polymerization reaction of the FibMA initiated by light activation. Rheological measurements were used to verify tandem crosslinking and characterize final DN hydrogel mechanical properties. Bioprocessing into hydrogel filaments was achieved using a 3D bioprinter with a custom-built co-axial extrusion nozzle. C2C12 cell-laden FibMA-fibrin hydrogel filaments were formed, and live/dead staining was used to evaluate the cell viability in 3D culture. Quantitative cell spreading and alignment was evaluated in DN hydrogel filaments and compared to bulk hydrogel constructs using f-actin fluorescence staining.

Results, Conclusions, and Discussions: FibMA-fibrin DN hydrogels exhibited rapid self-assembly of fibrin through supramolecular interactions, which enabled extruding stable filaments with a 3D bioprinter. The covalent FibMA network interacted with the fibrin to substantially enhance the viscoelastic mechanical properties of the DN filaments, including the strength and modulus. C2C12 cell viability was high in the DN filaments after 2 weeks in 3D culture. The fibrin microstructure enhanced cell morphogenesis within the dense FibMA network, facilitating accelerated uniaxial cellular reorientation and alignment in the filaments through contact guidance cues. The morphological orientation of encapsulated C2C12 cells after one week was highly aligned with the filament axis whereas bulk gels exhibited randomly orientated C2C12 cells. We conclude that the unique microstructure of the FibMA-fibrin DN hydrogels combined with their high strength and the anisotropy of the filament structures support C2C12 alignment and maturation towards a myogenesis trajectory.

Acknowledgements (Optional): We acknowledge the financial support of the Israel Innovation Authority, the Israel Science Foundation and the Israel Council for Higher Education. We thank the Ariane de Rothschild Women Doctoral Program of the Rothschild Caesarea Foundation for their generous support.