Tuning cellular response by modular design of bioactive domains in collagen

Abstract

Collagen's ability to direct cellular behavior suggests that redesigning it at the molecular level could enable manipulation of cells residing in an engineered microenvironment. However, the fabrication of full-length collagen mimics of specified sequence de novo has been elusive, and applications still rely on material from native tissues. Using a bottom-up strategy, we synthesized modular genes and expressed recombinant human collagen variants in Saccharomyces cerevisiae. The resulting biopolymers contained prescribed cell-interaction sites that can direct and tune cellular responses, with retention of the important triple-helical self-assembled structure. Removal of the native integrin-binding sites GROGER, GAOGER, GLOGEN, GLKGEN, and GMOGER in human collagen III yielded collagen that did not support adhesion of mammalian cells. Introduction of GFOGER sequences to this scaffold at specified locations and densities resulted in varying degrees of cellular attachment. The recruitment of focal adhesion complexes on the different collagens ranged from a 96% reduction to a 56% increase over native collagen I. Adhesion to the GFOGER-containing variants was entirely dependent and partially dependent on the β1 and α2 subunits of integrin, respectively, with cell adhesion on average reduced by 86% with anti-β1 and 38% with anti-α2 integrin antibody incubation. Results support the importance of local context in collagen-cell interactions. The investigation demonstrates the flexibility of this approach to introduce targeted changes throughout the collagen polymer for producing fully-prescribed variants with tailored properties.