(718h) Tuning Bulk Hydrogel Degradation By Simultaneous Control of Proteolytic Cleavage Kinetics and Hydrogel Network Architecture

Hydrogel-based materials that mimic the native extracellular matrix (ECM) have found uses as tissue engineering scaffolds, injectable carriers for stem cell therapies, and three-dimensional (3D) cell culture platforms. These synthetic systems are engineered to recapitulate critical aspects of the biochemical and biophysical cues provided by the native cellular microenvironment. In engineered ECM materials, properties such as stiffness, viscoelasticity, adhesive ligand interactions, and growth factor presentation have been shown to regulate cellular behaviors including proliferation, differentiation, and migration. Embedding cells within 3D hydrogels enables recapitulation of key aspects of the native ECM that are absent from conventional two-dimensional (2D) culture. A particular advantage provided by 3D culture is the ability to study matrix degradation and remodeling. While 2D culture permits unrestricted spreading, proliferation, and migration, matrix remodeling of 3D hydrogels is required to facilitate these same behaviors, which are crucial to processes such as blood vessel sprouting and cancer metastasis. Accordingly, several elegant approaches have been employed to render hydrogels degradable by cell-secreted proteases. However, existing hydrogel systems are limited in their ability to simultaneously and quantitatively tune two aspects of hydrogel degradability: cleavage rate (the rate at which individual chemical bonds are cleaved) and degraded hydrogel architecture (the network structure during degradation). Whereas these two parameters are commonly combined into a qualitative “degradability” term, we have developed a single hydrogel system in which both bond cleavage rate and degraded hydrogel architecture can be quantitatively modulated. Using standard peptide engineering approaches, we alter the proteolytic kinetics of the polymer cleavage rate to tune gel degradation time from less than 12 hours to greater than 9 days in response to treatment with the cell-secreted enzyme urokinase plasminogen activator (uPA). Independently, we vary the functionality of multi-arm poly(ethylene glycol) (PEG) crosslinkers to achieve network architectures that initially have identical molecular weight between crosslinks, but upon degradation are designed to release between 5% to 100% of the polymer. To confirm the biological relevance of both parameters, we cultured murine brain microvascular endothelial cells that secrete uPA in hydrogels with varying bond cleavage rates and degraded hydrogel architecture. Strikingly, formation of vascular-like structures by the endothelial cells is regulated both by bond cleavage rate and by degraded hydrogel architecture. These results indicate that degradation kinetics and degradation architecture are separate, quantitative design variables that are both critical for cell biology. Our strategy to fine-tune different aspects of hydrogel degradability has applications in cell culture, regenerative medicine, and drug delivery.