Development of Phase-Separating Microfiber Network Hydrogels to Promote In Vitro Vascularization

Engineered vascularized tissues in vitro exhibit the potential for transplantation therapy and disease modeling. Despite efforts to design hydrogels as cell culture platforms for in vitro vascularization, development of vascularized tissues recapitulating the natural structures and functions remains difficult due to a poor understanding of the relationships between the matrix microstructures and tube formation of endothelial cells. Herein, we developed microfiber network hydrogels with microporous structures by controlling the liquid-liquid phase separation (LLPS) of proteins and matrix structures in hydrogels. Extracellular matrix protein gelatin was modified with hydrogen-bonding moieties and mixed with hyaluronic acid sodium salt to form microfiber network structures. Gelatin gelation and hyaluronic acid sodium salt dissolution led to the formation of a microporous microfiber network hydrogel formation. Matrix structures of hydrogels were modified by controlling LLPS that affects endothelial cell tube formation. Vascularization was improved using laminin peptides and coculturing with mesenchymal stem cells. Overall, our approach exhibits the potential to induce in vitro vascularization for regenerative medicine and disease modeling applications.