3D-bioprinting is a promising technique to mimic the complex anatomy of natural tissues, as it comprises a precise and gentle way of placing bioinks containing cells and hydrogel. Although hydrogels expose an ideal growth environment due to their extracellular matrix (ECM)-like properties, high water amount and tissue like microstructure, they lack mechanical strength and possess a diffusion limit of a couple of hundred micrometers. Integration of electrospun fibers could hereby benefit in multiple ways, for instance by controlling mechanical characteristics, cell orientation, direction of diffusion and anisotropic swelling behavior. The aim of this study was to create an advanced ECM-biomimicking scaffold material for tissue engineering, which offers enhanced diffusion properties. PCL bulk membranes were successfully electrospun and fragmented using a cryo cutting technique. Subsequently, these short single fibers (<400µm in length and ∼5-10µm in diameter) were embedded in an agarose-based hydrogel after hydrophilization of the short single fibers by O₂plasma treatment. Fiber-filled bioinks exhibit significantly improved biomolecule diffusion (>500µm), swelling properties (20%-60% of control), and higher mechanical strength, while its viscosity (5-30 mPas*s) and gelation kinetics (28 °C) remained almost unaffected. The diffusion tests indicate a high level of size selectivity, which can be utilized for targeted biomolecule transport in the future. Finally, applying 3D-bioprinting technology (drop-on-demand vs. microextrusion) a print setting dependent post-dispensing orientation of the fibers could be induced, which ultimately paves way for the fabrication of metamaterials with anisotropic material properties. As expected, the fiber-filled bioink was found to be non-cytotoxic in cell culture trials using HUVECs and HepG2 (>80% viability). In summary, microfiber integration holds great promise for 3D-bioprinting of tissue percursors with advanced metamaterial properties and thus offers high applicability in various fields of research, such asin-vitrotissue models, tissue engineered implants or cultivated meat.

Annabelle Neuhäusler

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