The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviour.

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonizationover time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaClduring the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of theenvironment.