Optimization of Freeze-FRESH Methodology for 3D Printing of Microporous Collagen Constructs

Freeform reversible embedding of suspended hydrogels (FRESH) is a layer-by-layer extrusion-based technique to enable three-dimensional (3D) printing of soft tissue constructs by using a thermo-reversible gelatin support bath. Suboptimal resolution of extrusion-based printing limits its use for the creation of microscopic features in the 3D construct. These microscopic features (e.g., pore size) are known to have a profound effect on cell migration, cell-cell interaction, proliferation, and differentiation. In a recent study, FRESH-based 3D printing was combined with freeze-casting in the Freeze-FRESH (FF) method, which yielded alginate constructs with hierarchical porosity. However, use of the FF approach allowed little control of micropore size in the printed alginate constructs. Herein, the FF methodology was optimized for 3D printing of collagen constructs with greater control of microporosity. Modifications to the FF method entailed melting of the FRESH bath before freezing to allow more efficient heat transport, achieve greater control on microporosity, and permit polymerization of collagen molecules to enable 3D printing of stable microporous collagen constructs. The effects of different freezing temperatures on microporosity and physical properties of the 3D-printed collagen constructs were assessed. In addition, finite element (FE) models were generated to predict the mechanical properties of the microporous constructs. Further, the impact of different micropore sizes on cellular response was evaluated. Results showed that the microporosity of 3D-printed collagen constructs can be tailored by customizing the FF approach. Compressive modulus of microporous constructs was significantly lower than the non-porous control, and the FE model verified these findings. Constructs with larger micropore size were more stable and showed significantly greater cell infiltration and metabolic activity. Together, these results suggest that the FF method can be customized to guide the design of 3D-printed microporous collagen constructs.