pH and Thrombin Concentration are Decisive in Synthesizing Stiff, Stable, and Open-Porous Fibrin-Collagen Hydrogel Blends without Chemical Crosslinker.

Fibrin-collagen hydrogel blends exhibit high potential for tissue engineering applications. However, it is still unclear whether the underlying crosslinking mechanisms are of chemical or physical nature. We hypothesize that chemical crosslinkers play a negligible role and that instead pH and thrombin concentration are decisive for synthetizing blends with high stiffness and hydrolytic stability. We use different fibrin-collagen formulations (pure and with additional transglutaminase) and investigate the blends' compaction rate, hydrolytic stability, compressive strength, and hydrogel microstructure. We examine the effect of thrombin concentration on gel compaction and observe the importance of pH control during synthesis. We reveal that transglutaminase impairs gel stability and deduce that fibrin-collagen blends mainly crosslink by mechanical interactions due to physical fibril entanglement as opposed to covalent bonds from chemical crosslinking. High thrombin concentrations and basic pH during synthesis reduce gel compaction and enhance stiffness and long-term stability. Scanning electron microscopy reveals a highly interpenetrating fibrous network with unique and interconnected open-porous microstructure. Endothelial cells proliferate on the blends and form a confluent monolayer. Our study reveals the underlying crosslinking mechanisms and presents enhanced fibrin-collagen blends with high stiffness, hydrolytic stability, and large and interconnected pores, findings that offer high potential for advanced tissue engineering applications. This article is protected by copyright. All rights reserved.