Creating protease-responsive hydrogels to generate cell-specific niches within matrices

About This Grant

PROJECT SUMMARY The inability to build physiologically relevant in vitro tissue models greatly limits both research capabilities and regenerative therapies. Hydrogels are frequently used to mimic the extracellular matrix (ECM) which surrounds cells in tissue, and the physical properties of these scaffolds can be tailored to individual cell types. These scaffolds are often made from polymers crosslinked by peptides that are substrates for cell-secreted proteases to enable the encapsulated cells to spread and migrate within the matrices. A challenge for these systems is that each cell type within a tissue can have a unique set of ideal matrix parameters. For instance, most tissues are highly vascularized, but endothelial network formation is optimized within matrices that are very soft, while other physiological processes, such as osteogenic differentiation, are typically optimized within stiffer, more highly crosslinked hydrogel matrices. This highlights the need for making hydrogels with specific niches for each cell type. To address this need, we propose fabricating scaffolds in which cell-specific protease activity creates tailored microenvironments around individual cell types. Each cell type expresses a unique combination of proteases, and we have developed novel methods to identify peptides that are specifically cleaved by individual cell types. We are also able to determine whether these peptides are cleaved near the surface of the cell or by soluble proteases that induce bulk matrix degradation. Using a "split-and-pool" peptide synthesis technique, we can generate more than 300 variants of protease-substrate peptides to tune the degradation rates to desired values. We hypothesize that hydrogels crosslinked with peptides with optimal spatiotemporal degradation kinetics will have increased biological performance over existing crosslinking peptides. We will test this hypothesis in two Aims: In Aim 1, we will use a split-and-pool synthesis technique to identify hydrogel crosslinking peptides whose degradation kinetics are optimized for either osteogenic differentiation of human mesenchymal stem cells (hMSCs) or vasculogenesis of human umbilical vein endothelial cells (hUVECs). We will also make peptides which are conjugated with chemically-labile bonds that will enable quantification of the fraction of crosslinks cleaved during culture, which will couple physiological behavior in gels to dynamic changes in hydrogel structure. In Aim 2, we will develop co-culture hydrogels that contain both hMSCs and hUVECs to identify a single peptide that supports both osteogenesis and vasculogenesis within hydrogels. This will pioneer the use of hydrogel crosslinking peptides to simultaneously promote multiple physiological processes within a single system. The proposed research plan combines biomaterial synthesis, analytical chemistry, and cell culture to develop a versatile platform that can be used across tissue systems to improve our ability to model tissues in vitro and regenerate them in vivo.