Multi-nozzle bioprinting of biomimetic tissue models with mechanical reinforcement and bioactive matrix remodeling

Abstract Replicating the complex mechanical and biological properties of native tissues remains a key challenge in three-dimensional bioprinting due to the limitations of single-nozzle systems. Here we present a multi-nozzle alternating bioprinting platform that addresses these problems by enabling precise control of mechanical and bioactive components’ composition and distribution. By alternating cell-laden bioinks with mechanically reinforcing inks, our method enables precise spatial control for fabricating complex, anisotropic tissue architectures. A tri-layer printing strategy, using heart valve leaflets as a demonstrative model, was developed. In detail, gelatin methacryloyl bioinks, incorporating with porcine aortic valve interstitial cells and bioactive substances (e.g. basic fibroblast growth factor, polyaspartic acid, or chondroitin sulfate) to support cell function, are alternated with pluronic F-127 diacrylate mechanical reinforcement inks. This approach enhanced mechanical integrity of the constructs while supporting collagen, proteoglycan, and elastin production. Crucially, the constructs’ mechanical robustness allowed direct cyclic mechanical stimulation during culture, further promoting tissue functional maturation and extracellular matrix remodeling. In vivo , the constructs showed excellent biocompatibility, with minimal calcification and favorable immune responses. This multi-material bioprinting platform enables the fabrication of tissue models that meet both structural and functional requirements, and can be adapted for a wide range of heterogeneous tissue and organ engineering applications, with the potential to significantly advance regenerative medicine.