Comb-assisted 3D bioprinting for highly aligned 3D muscle bioconstructs with enhanced cellular mechanotransduction

Mechanotransduction is essential for tissue regeneration, yet conventional 3D bioprinting methods often fail to provide the necessary mechanical stimuli, especially when using low-viscosity bioinks that produce insufficient shear stress. This shortcoming limits the ability to properly stimulate cells during bioprinting, affecting tissue formation and function. To address this issue, we developed a comb-assisted bioprinting technique aimed at enhancing mechanotransduction by increasing shear stress on the bioink. We used a 3 wt% collagen bioink laden with C2C12 myoblasts and human adipose-derived stem cells (hASCs), incorporating a comb-shaped microstructure attached to the bioprinter nozzle. By varying parameters such as bristle gap of comb-structure and nozzle moving speed, we were able to exert proper shear stress during the printing process. The comb-assisted bioprinting process led to significant mechanical stimulation, improving cell alignment and promoting the expression of mechanosensitive genes. Additionally, the method enhanced myotube formation, myosin heavy chain (MHC) alignment, and increased the expression of late-stage myogenesis-related genes. These outcomes contributed to better cellular alignment and superior myogenic activity in the engineered muscle constructs compared to conventional bioprinting methods. This method represents a promising advancement for the fabrication of engineered muscle tissues, addressing a key limitation of traditional bioprinting techniques.