FRESH bioprinting technology for tissue engineering - the influence of printing process and bioink composition on cell behavior and vascularization.

The rapid and tailored biofabrication of natural materials is of high interest for the field of tissue engineering and regenerative medicine. Scaffolds require both high biocompatibility and tissue-dependent mechanical strength to function as basis for tissue-engineered implants. Thus, natural hydrogels such as fibrin are promising but their rapid biofabrication remains challenging. Printing of low viscosity and slow polymerizing solutions with good spatial resolution can be achieved by freeform reversible embedding of suspended hydrogels (FRESH) bioprinting of cell-laden natural hydrogels. In this study, fibrin and hyaluronic acid were used as single components as well as blended ink mixtures for the FRESH bioprinting. Rheometry revealed that single materials were less viscous than the blended bioink showing higher values for viscosity over a shear rate of 10-1000 s. While fibrin showed viscosities between 0.1624 and 0.0017 Pa·s, the blended ink containing fibrin and hyaluronic acid were found to be in a range of 0.1-1 Pa·s. In 3D vascularization assays, formation of vascular structures within the printed constructs was investigated indicating that the printing process did not harm cells and allowed formation of vasculature comparable to moulded control samples. Best values for vascularization were achieved in bioinks consisting of 1.0% fibrin-0.5% hyaluronic acid. The vascular structure area and length were three times higher compared to other tested bioinks, and structure volume as well as number of branches revealed almost four times higher values. In this study, we combined the benefits of the FRESH printing technique with in vitro vascularization, showing that it is possible to achieve a mechanically stable small-scale hydrogel construct incorporating vascular network formation.