Electrohydrodynamic bioprinting-induced orientation of cell-laden fibrin-alginate hydrogel for highly-aligned skeletal muscle constructs

Bioprinting provides an unparalleled tool for engineering living tissue constructs that mimic the structural organization of native skeletal muscles. However, it remains a challenge for existing bioprinting strategies to recapitulate the highly aligned cellular architectures inside skeletal muscles, primarily due to low printing resolution and limited capability for in situ microenvironmental regulation. Here, we propose to employ the electrical force during the electrohydrodynamic (EHD) bioprinting process to induce the in situ orientation of cell-laden fibrin-alginate hydrogel, which provides nanostructural guidance to the encapsulated cells for the formation of highly aligned skeletal muscle constructs. It was observed that the randomly distributed fibrin protofibril aggregates gradually elongated into uniformly aligned nanofibers at the Taylor cone stage as the applied voltage increased to 3 kV. The oriented fibrin nanofibers further direct in situ cellular alignment along the EHD bioprinting trajectory, facilitating the freeform fabrication of parallelly or circumferentially aligned muscle tissue constructs in vitro. The addition of conductive polymers into the fibrin-alginate hydrogel endows the EHD-bioprinted living constructs with muscle-specific conductivity and cellular organization, which promote myotube differentiation and maturation. The resultant aligned and conductive muscle constructs promoted in situ muscle regeneration and restored lost muscle functions at the defect regions in vivo. The presented EHD bioprinting strategy for fibrin-alginate hydrogel provides a versatile and simple platform to freely fabricate conductive, living tissue constructs with designer cellular alignments.