Freeze-FRESH: A 3D Printing Technique to Produce Biomaterial Scaffolds with Hierarchical Porosity.

Tissues are organized in hierarchical structures comprised of nanoscale, microscale, and macroscale features. Incorporating hierarchical structures into biomaterial scaffolds may enable better resemblance of native tissue structures and improve cell interaction, but it is challenging to produce such scaffolds using a single conventional scaffold production technique. We developed the Freeze-FRESH (FF) technique that combines FRESH 3D printing (3DP) and freeze-casting to produce 3D printed scaffolds with microscale pores in the struts. FF scaffolds were produced by extrusion 3DP using a support bath at room temperature, followed by freezing and lyophilization, then the FF scaffolds were recovered from the bath and crosslinked. The FF scaffolds had a hierarchical pore structure from the combination of microscale pores throughout the scaffold struts and macroscale pores in the printed design, while control scaffolds had only macroscale pores. FF scaffolds frozen at -20 °C and -80 °C had similar pore sizes, due to freezing in the support bath. The -20 °C and -80 °C FF scaffolds had porous struts with 63.55% ± 2.59% and 56.72% ± 13.17% strut porosity, respectively, while control scaffolds had a strut porosity of 3.15% ± 2.20%. The -20 °C and -80 °C FF scaffolds were softer than control scaffolds: they had pore wall stiffness of 0.17 ± 0.06 MPa and 0.23 ± 0.05 MPa, respectively, compared to 1.31 ± 0.39 MPa for the control. The FF scaffolds had increased resilience in bending compared with control. FF scaffolds supported MDA-MB-231 cell growth and had significantly greater cell numbers than control scaffolds. Cells formed clusters on the porous struts of FF scaffolds and had similar morphologies as the freeze cast scaffolds. The FF technique successfully introduced microscale porosity into the 3DP scaffold struts to produce hierarchical pore structures that enhanced MDA-MB-231 growth.