Synthetic Surrogates of Collagen-Rich Microenvironments: Integrating Modular Bioactive Fibrillar Structure and Tunable Viscoelasticity via Multifunctional Assembling Peptides

Collagen imparts structure, viscoelasticity, and bioactivity to the extracellular matrix (ECM) with variance between organs and between healthy and diseased states. Synthetic mimics of collagen-rich tissues remain a need in applications, from biological studies to regenerative medicine, for parsing and controlling these properties. We designed multifunctional collagen mimetic peptides (mfCMPs) that self-assemble into triple helices and fibrils and contain integrin binding motifs: GFOGER, a binding site within intact collagen I, and RGD, a cryptic binding site available within denatured collagen I. These mfCMPs are incorporated into hydrogel-based synthetic ECMs to impart collagen-like hierarchical structures, viscoelasticity, and bioactivity with modularity. We establish innovative methods for imaging the resulting nano- and micro-structures within the hydrogel using super-resolution microscopy. The physically assembled mfCMPs impart tunable, concentration-dependent viscoelasticity within otherwise elastic, covalently cross-linked hydrogels, exhibiting relaxation half times over orders of magnitude and similar to soft tissues. Notably, breast cancer cells encapsulated and cultured in synthetic ECMs with a bioactive fibrillar structure and viscoelastic properties formed large, growing spheroids. Overall, these modular building blocks provide an innovative tool for creating fully synthetic surrogates of collagen-rich microenvironments, aiding both fundamental and translational biological studies and providing a framework to imbue tunable viscoelasticity to synthetic ECMs.