(314e) Dynamic Stiffening of Poly(ethylene glycol)-Based Hydrogels to Direct Valvular Interstitial Cell Phenotype in a Three-Dimensional Environment

Valvular interstitial cells (VICs) are the primary cell type within the aortic valve and are active regulators in the progression of valve disease. Extended activation of the VICs to a myofibroblast phenotype can lead to valve fibrosis and calcification. Environmental stiffness is a critical determinant of myofibroblast activation. While there has been much progress in controlling VIC activation by the introduction of mechanical and biochemical cues in two-dimensional cell culture systems, there have been few attempts to direct VIC phenotypes with matrix stiffness in three-dimensional environments. Here, we encapsulate VICs in poly(ethylene glycol)-based hydrogels crosslinked with a matrix metalloprotease-degradable peptide to manipulate their phenotype with stiffness cues in a three-dimensional environment. We found that only VICs encapsulated in the softest hydrogels (Young’s modulus, E = 0.24 kPa) exhibited alpha smooth muscle actin stress fibers, a hallmark of the myofibroblast phenotype. To decouple the effect of cell morphology from the matrix stiffness, a dynamic materials platform was developed to increase the modulus of cell-laden hydrogels using a light-initiated, cytocompatible thiol-ene chemistry. VICs were permitted to spread and elongate in 0.24 kPa hydrogels before gels were stiffened to 1.2 kPa or 13 kPa. High cell viability was observed after stiffening. Further, by increasing the matrix stiffness, the VICs could be directed into a deactivated phenotype. This result is in direct contrast with the response of VICs to modulus in two-dimensional systems, suggesting that the dimensionality of a cell’s microenvironment influences the cellular response to mechanical cues. Future work will explore the impact of degradability of the gel on VIC phenotype by comparing activation levels in stiffened gels with or without a matrix metalloprotease-degradable crosslinker.

Support for this work provided by the NIH (5R01HL089260 and Pharmaceutical Biotechnology training grant) and HHMI.