Poster O7 - Engineering the mechanical and biological properties of biomacromolecular granular hydrogel scaffolds via microgel packing

Introduction: Granular hydrogel scaffolds (GHS) have been developed to overcome the structural limitations of bulk (nanoporous) hydrogels in tissue engineering and regeneration. GHS are fabricated by placing hydrogel microparticles (HMP) in close contact (packing), followed by physical and/or chemical interparticle bond formation. Various techniques have been used for confining the HMP in close-packed conditions, including centrifugation and compression. Studies have shown the effect of centrifugal speed on the porosity of GHS with different HMP sizes and/or stiffness. However, the effects of packing on the physical and biological properties of gelatin methacryloyl (GelMA) GHS are unexplored.

Materials and

Methods: GelMA, synthesized by methacrylate modification of gelatin, was used to fabricate uniformly sized HMP via a step-emulsification microfluidic device (Fig. 1a). To investigate the effect of packing on GelMA GHS porosity, mechanical properties, in vitro cell viability and migration, and in vivo response, GelMA HMP were packed at different centrifugal forces and durations. Fluorescence microscopy was used to analyze the void fraction and the median pore equivalent diameter of GHS (Fig. 1b). In addition, mechanical characterization of the GHS was conducted via compression and frequency sweep tests. GHS was cultured with NIH/3T3 fibroblast and MDA-MB-231 breast cancer cells or subcutaneously implanted in mice for in vitro or in vivo characterizations, respectively.

Results, Conclusions, and Discussions: Increasing centrifugation time/force resulted in a high degree of HMP packing, decreasing the void fraction and median pore diameter. Increased HMP contact area at the highly packed condition resulted in higher GHS compressive and storage moduli. The cell viability and metabolic activity in GelMA GHS were analyzed for 7 days. Metabolic activity decreased on day 7 as the packing increased, which might be a result of higher cell density per volume when pore diameter and void fraction decreased (Fig. 1c). The migration length of cells within GHS was analyzed three days after seeding, and a decrease in migration length was observed by increasing the packing (Fig. 1d). Additionally, MDA-MB-231 triple-negative breast cancer cells spread more on the surface of HMP in loosely packed GHS compared with the highly packed counterpart (Fig. 1e). In vivo subcutaneous implantation conducted on the GHS showed a lower cell migration and tissue integration within the highly packed GHS. Harvested tissue was stained with a cluster of differentiation 31 (CD31), CD68, CD86, CD206, and α-smooth muscle actin (α-SMA) markers for analyzing the effect of packing on the invasion of endogenous cells and vessel formation within GHS. A lower cell coverage was observed in the highly packed GHS compared with the loosely packed one. In conclusion, we engineered GelMA GHS porosity by applying a centrifugal force to induce HMP deformation and showed how mechanical and biological responses were affected by the packing. This research is a step forward in engineering GelMA GHS via external stimuli for tissue engineering.

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