Combining Supramolecular and Covalent Chemistry to Form Reinforced Fibrillar Network Hydrogels From Fibrinogen Derivatives

Fibrin hydrogels provide a highly supportive environment for three-dimensional cell growth and morphogenesis but are limited by insufficient stability to maintain their shape and mechanical integrity. To address this limitation, we developed a reinforced fibrin hydrogel incorporating photo-crosslinked methacrylated fibrinogen (FibMA). These interpenetrating polymer network (IPN) hydrogels were formed by thrombin-mediated activation of fibrinogen to initiate supramolecular fibrin assembly (fibrillogenesis), followed by light-activated covalent crosslinking of FibMA (photopolymerization). The unique interaction between the supramolecular and covalent chemistries, in tandem, resulted in a reinforced fibrillar network (RFN) architecture with exceptional control over mechanical properties. Precise control over the dynamic viscoelastic moduli of the FibMA-fibrin IPN hydrogel (FibMAtrx) was achieved by delaying the initiation of the photochemistry, thereby increasing the thrombin reaction time (TRT). Following covalent crosslinking, FibMAtrx exhibited more than a fivefold increase in dynamic viscoelastic moduli, depending on TRT and hydrogel composition. Fibroblasts encapsulated within FibMAtrx IPNs exhibited high viability and extensive morphogenesis, with quantitative cell spreading governed primarily by FibMA content rather than hydrogel stiffness. Cell-mediated hydrogel shrinkage after 1 week of culture (i.e., compaction) was substantially reduced in FibMAtrx IPNs, whereas fibrin single-network hydrogels compacted to a fraction of their original size. Together, these findings demonstrate the potential of proteinaceous reinforced fibrillar IPN hydrogels derived from fibrinogen and FibMA to support cell growth, enable tunable viscoelastic properties, resist compaction and enzymatic degradation, and maintain shape integrity in cell-laden fibrillar protein hydrogels.