Composite Alginate\nDialdehyde-Gelatin (ADA-GEL) Hydrogel\nContaining Short Ribbon-Shaped Fillers for Skeletal Muscle Tissue\nBiofabrication

Skeletal muscle tissue can be severely damaged by disease\nor trauma\nbeyond its ability to self-repair, necessitating the further development\nof biofabrication and tissue-engineering tools for reconstructive\nprocesses. Hence, in this study, a composite bioink of oxidized alginate\n(ADA) and gelatin (GEL) including cell-laden ribbon-shaped fillers\nis used for enhancing cell alignment and the formation of an anisotropic\nstructure. Different plasma treatments combined with protein coatings\nwere evaluated for the improvement of cell adhesion to poly(lactic-<i>co</i>-glycolic acid) (PLGA) ribbon surfaces. Oxygen plasma\nactivation of 30 W for 5 min showed high immobilization of fibronectin\nas a protein coating on the PLGA ribbon surface, which resulted in\nenhanced cell adhesion and differentiation of muscle cells. Furthermore,\nthe effect of various concentrations of CaCl<sub>2</sub> solution,\nused for ionic cross-linking of ADA, on ADA-GEL physical and mechanical\nproperties as well as encapsulated C2C12 cell viability and proliferation\nbehavior was investigated. The pore area was measured via two approaches,\ncryofixation and lyophilization, which, in accordance with degradation\ntests and mechanical analysis, showed that 60 mM CaCl<sub>2</sub> concentration\nis the optimum range for cross–linking of the formulation of\nADA 2.5%w/v–GEL 3.75%w/v. These cross-linked hydrogels showed\na compression modulus of 11.5 kPa (similar to the native skeletal\nmuscle tissue), a high viability of C2C12 muscle cells (>80%),\nand\na high proliferation rate during 7 days of culture. Rheological characterization\nof the ADA-GEL composite hydrogel containing short fillers (100 μm\nlong) showed its suitability as a bioink with shear-thinning and flow\nbehavior compared to ADA-GEL.