Directional biofabrication using collagen-based decellularized extracellular matrix bioink for multi-layered cellular...

Cellular alignment is a fundamental characteristic of tissues that enables them to perform specific functions effectively. In this study, we introduce a novel biofabrication approach to achieve cellular alignment across multiple layers in specific intended directions using collagen-based decellularized extracellular matrix (ECM) bioink via extrusion-based 3D bioprinting technology. Through both experimental validation and mathematical modeling, we demonstrate that our approach significantly enhances the alignment of collagen microfibers and encapsulated cells in the intended directions across multiple layers. We performed a comprehensive analysis of collagen microfiber orientation, top-seeded cell alignment, and encapsulated cell positioning to fabricate various tissue types with the desired cellular organization. Using this innovative technique, we successfully produced three types of cellularly aligned muscle tissue patches-smooth, skeletal, and cardiac muscle-each exhibiting functional characteristics, as confirmed by myogenic differentiation through RT-PCR. Additionally, we created a cellularly aligned endothelial layer, a transparent corneal tissue patch, and neural tissue. Notably, when osteoblast-like cells encapsulated in collagen-based decellularized ECM bioink were printed onto prefabricated PCL-silk constructs, we observed significant bone mineralization, indicating effective neobone formation. Our methodology effectively addresses the challenges associated with fabricating cellularly aligned tissues, advancing the field of tissue engineering and opening new avenues for practical applications in regenerative medicine.