3D Printed Microporous Collagen Constructs Using the Freeze-FRESH Methodology

Abstract

Scaffold microporosity is known to play a critical role in governing cellular response including cell migration, proliferation, and tissue-specific differentiation. While fabrication approaches such as solvent leaching and freeze-casting are commonly used for the generation of microporous biomaterial scaffolds, these methods provide little control over scaffold geometry and creation of complex tissue structures. Extrusion-based 3D printing, an additive manufacturing method, is a highly versatile layer-by-layer printing approach that allows for the fabrication of easily customizable 3D scaffolds with complex architecture using a vast selection of polymeric bioinks. These 3D printed constructs are porous at the macroscale, but achieving microporosity (i.e., < 100 μm) in printed constructs is challenging due to the sub-optimal resolution of the extrusion-based printing method. A recent study using alginate inks introduced a new fabrication technique termed Freeze-FRESH(FF)that combines extrusion printing and freeze-casting approaches to generate 3D constructs with hierarchical microporosity. However, the porosity of the resultant alginate constructs was comparable despite changing the freezing temperature. In the current study, the FF method was modified to print collagen constructs with greater control of microporous architecture. Highly concentrated collagen type I ink was used to 3D print collagen constructs using the freeform reversible embedding of suspended hydrogels (FRESH)approach. Modification of the FF technique entailed melting of the FRESH bath post printing via incubation at 37 °C followed by freezing and lyophilization to allow for collagen gelation and better heat transport during the freezing process. The effect of freezing temperature on micropore size, swelling degree, degradation, and mechanical properties of printed constructs was assessed. Finite element (FE) models were generated to predict the mechanical properties of microporous scaffolds. In addition, the effect of microporosity on Saos-2 cell morphology, proliferation, infiltration, and ALP activity was evaluated. Results from the study showed that freezing temperature effectively modulated micropore size and that constructs with larger micropore size were more stable. Microporosity had no effect on swelling ratio yet caused a decrease in mechanical properties of collagen constructs; FE models confirmed experimental results. Cell metabolic activity and infiltration was enhanced in microporous constructs with larger pore size, yet there was no effect on cell morphology, and ALP activity. In conclusion, the modified FF technique can be effectively used to fabricate hierarchically porous 3D collagen constructs.