UC San Diego

Tissue-specific elastic modulus (E), or 'stiffness,' arises from developmental changes in the extracellular matrix (ECM) and suggests that progenitor cell differentiation may be optimal when physical conditions mimic tissue progression. For the myocardium, changes in extracellular matrix over time results in a ̃10-fold stiffening in the chicken embryo. To mimic this temporal stiffness change in vitro, thiolated hyaluronic acid (HA- SH) hydrogels were crosslinked with poly(ethylene glycol) diacrylate, and their dynamics were modulated by changing crosslinker molecular weight and component compositions. With the hydrogel appropriately tuned to stiffen as heart muscle does during development, embryonic cardiomyocytes grown on collagen-coated HA hydrogels exhibited a 3-fold increase in mature cardiac specific markers and form up to 60% more maturing muscle fibers than they do when grown on compliant but static polyacrylamide hydrogels over 2 weeks. While active mechanotransduction aided maturation, the specific proteins responsible for responding to time- dependent stiffness remain unknown. In order to assess matrix-mediated mechanotransduction, the expression and phosphorylation state of 800 protein kinases was examined for embryonic cardiomyocytes plated on matrices with either dynamic or static cardiac tissue-specific stiffness. Microarray analysis of protein kinases showed differential expression as a function of mechanics; many cardiogenic pathways exhibited time-dependent up- regulation on dynamic versus static matrices, including PI3K/Akt and p38 MAPK, while GSK3[beta], a known inhibitor of cardiomyocyte maturation was down regulated. Though improved cardiomyocyte maturation was observed in vitro, host interactions, matrix polymerization, and the stiffening kinetics remain uncertain in vivo, and each plays a critical role in therapeutic applications using HA -SH. Subcutaneously injected HA-SH hydrogels showed minimal systemic immune response and host cell infiltration and exhibited time-dependent porosity and stiffness changes at a rate similar to hydrogels polymerized in vitro. When injected intramyocardially, visible granulomas and macrophage infiltration were present 1 month post-injection, likely due to reactive thiol groups. Altogether these data demonstrate the development of a novel hydrogel system that displays dynamic developmental cues in order to enhance embryonic cardiomyocyte maturation in vitro; however, the in vivo applicability of this material in vascularized tissue appears limited