Structure-functionality relationship of collagen-fibrin interpenetrating hydrogels for engineered tumor-stroma models

Collagen and fibrin are two major extracellular matrix (ECM) proteins involved in tumorigenesis. During cancer progression, collagen and fibrin undergo sequential and concurrent polymerization and degradation, altering ECM structure and functional properties, which impact cell behavior and drug resistance. While engineered tumor models are recently emerging, including 3D printed tissue models and microphysiological systems (MPS), the interaction of these two proteins and its impact on the structure-functionality are largely overlooked. A critical knowledge gap on the structure-functionality relationship of collagen-fibrin interpenetrating hydrogels limits reliable reconstitution of the ECM of the tumor-stroma microenvironment. To address this, we characterized the fibrillar microstructure and biomechanical properties (e.g., diffusivity, Young's modulus, and cell-derived contraction) of collagen-fibrin interpenetrating hydrogels while varying the protein ratios and sequence of polymerization. The results show that the pore size of the composite matrices decreases as fibrin content increases. This structural change is correlated to the decreased diffusivity, which can be partially recovered after fibrin depletion. In contrast, Young's modulus only increases at lower fibrin content and decreases with increases in fibrin content, even below collagen-only levels. Cell-derived contraction is well correlated with these Young's modulus changes. Confocal microscopy analysis shows that collagen-fibrin gels have distinctly different microstructure depending on polymerization sequence, which highlights how collagen-fibrin interactions during polymerization shape matrix properties, with important implications for the design of engineered tumor models. STATEMENT OF SIGNIFICANCE: Engineered tumor models are emerging to recapitulate the complexity of human tumors with unprecedented cellular and molecular resemblance. Despite recent advancements, efforts to recreate the fibrin deposition caused by the extravascular coagulation due to leaky tumor vasculature is lacking. Furthermore, its impact on the structure-functionality relation of the tumor-stroma tissues poses a significant knowledge gap to reliably reconstitute biomaterials for engineered tumor models. To address this gap, we report how composition and polymerization conditions influence the microstructure and biomechanical properties of collagen-fibrin interpenetrating hydrogels. We show that both the collagen and fibrin concentration and polymerization sequence impact the interpenetrating microstructure and determine the diffusivity, Young's modulus, and contraction index of the hydrogel. These results are important by providing the quantitative knowledge-base for designing and analyzing biomaterials for new and innovative engineered tumor models.