3D in vitro model of skeletal muscle development using stiffening silk biomaterials

Hydrogels incorporating silk protein primed with bioactive peptides have been successfully used to study the cellular processes underlying differentiation of skeletal muscle. However, previous systems were limited due to their static nature – their mechanical properties are fixed. Recently, we demonstrated a new silk hydrogel crosslinked with tyramine-substituted silk fibroin that stiffened over time at controllable rates. The programmable stiffness of these hydrogels makes them attractive for modeling the changes in tissue-level stiffness that are associated with musculoskeletal development, or following injury. We will modify these hydrogels to incorporate decellularized muscle extracellular matrix (ECM), obtained through a recently established decellularization protocol. Our preliminary data suggest coupling ECM to our silk matrices can be used to further fine-tune the stiffening, enabling highly controllable and distinct mechanical and matrix protein gradients within the same gel, by spatially varying the amount and type of ECM mixed in with the silk precursors. A silk-ECM hydrogel has not previously been developed. Our central hypothesis is that dynamically stiffening hydrogels with highly tunable mechanical and biochemical characteristics can recapitulate key aspects of the myogenic environment more effectively than existing engineered systems, and as a result, will improve our understanding of the process to enable better control of the therapeutic potential of myogenically differentiating iPSCs for regenerating skeletal muscle. We will develop hydrogels as novel in vitro systems to explore the impacts of dynamic stiffness on myogenesis of iPSCs. We will test our hypothesis using two specific aims. The first aim will be to determine how evolving stiffness in 3D hydrogels impacts iPSC myogenesis. The second aim will be to develop a biochemically functionalized and mechanically dynamic silk-ECM hydrogel for generation of skeletal muscle from iPSCs. Completion of these aims will enhance our understanding of the regulators of skeletal muscle development and the impact of dynamic substrate stiffness and matrix composition on stem cell differentiation, with the ultimate goal of therapeutically targeting these mechanisms to regenerate skeletal muscle using stem cells. 3D hydrogels can be further used to investigate the processes that regulate development, aging, injury, and disease of skeletal muscle.