In this work, the novel bioink composed of alginate and polyethyleneimine were prepared with different concentrations of alginate and polyethyleneimine (PEI) and used to measure printability, rheological behavior, and cellular functions. The results indicated that viscosity tends to increase about 1.3 times with increasing the alginate and PEI content, which gradually affected structural fidelity. The presence of PEI can create a bioink that is partially gelled with higher storage modulus, high shape fidelity, and printability than alginate. Increasing in the mechanical strength and decreasing in the swelling rate for alginate/PEI bioink demonstrated the strong effect of PEI on the enhancing of structure rigidity in the alginate‐based bioinks. Finally, the cell viability and cell proliferation of the 3D alginate/PEI scaffolds were examined using live/dead and 40,6‐diamidino‐2‐phenylindole staining to select the best bioink formulation. Fibroblast‐laden bioink of 6% alginate and 7% PEI indicated the high proliferation of the printed cells via increasing in hydrophilicity of the surface. The in‐vivo evaluations on the wound rat model indicated that the 3D scaffold effectively accelerated the wound healing process and promoted tissue regeneration by activation of vimentin along with cytokeratin as well as collagen synthesis. Hair follicles started to form at Day 7 in alginate/PEI scaffold.
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