Comparative evaluation of recombinant thrombin and fibrin hydrogel systems for extracellular vesicle delivery†

Abstract

Extracellular vesicles (EVs) represent promising therapeutic agents in regenerative medicine due to their capacity to modulate cellular functions through cargo transport. However, their clinical effectiveness is limited by rapid systemic clearance and degradation, constraints potentially addressed through encapsulation within biocompatible hydrogel matrices. This study evaluates a novel fibrin hydrogel formulation derived from recombinant thrombin (Recothrom®) and residual fibrinogen present in purified exosome product (PEP) as an alternative to conventional fibrin sealant (TISSEEL®). We conducted comprehensive analyses of physicochemical properties, release kinetics, and cellular responses across four distinct hydrogel formulations: two conventional fibrin constructs with elevated fibrinogen-to-thrombin ratios (1) fibrin hydrogel with PEP supplementation (FG/T + PEP) and (2) non-supplemented fibrin hydrogel control (FG/T), and two Recothrom®-based variants with reduced fibrinogen-to-thrombin ratios (3) Recothrom® 25 units per ml + PEP (hrT25/PEP) and (4) Recothrom® 50 units per ml + PEP (hrT50/PEP). The Recothrom®-based formulations demonstrated significantly reduced mechanical stiffness characterized by thinner, more extensively branched fiber network architecture and decreased pore sizes. Although EV release dynamics were comparable across all groups, these structural characteristics facilitated enhanced cellular functionality promoting endothelial cell proliferation, upregulation of endothelial-specific markers, and improved fibroblast spreading, directional alignment, contractility, and type I collagen (COL-1) synthesis. These findings establish Recothrom®-based hydrogels as superior microenvironments for promoting angiogenesis and fibroblast-mediated extracellular matrix production, with significant implications for therapeutic EV delivery in tissue engineering applications.