Uncoupled investigation of scaffold modulus and mesh size on smooth muscle cell behavior

Although scaffold material properties are known to critically impact cell behavior, it has proven difficult to correlate specific cell responses to isolated scaffold parameters, inhibiting rational design of scaffold material properties. The aim of this study was to validate a systematic approach for evaluating the influence of initial scaffold modulus and mesh size on cell extracellular matrix (ECM) deposition and phenotype. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were selected for this study because of their tunable material properties. Following screening of six distinct PEGDA hydrogels, three formulations were identified which permitted uncoupled investigation of scaffold mesh size and modulus within the target incremental modulus range of approximately 100-300 kPa. Smooth muscle cells (SMCs) were encapsulated within these three formulations, and cell ECM deposition and phenotype were evaluated following 21 days of culture. Although elastin content appeared to be correlated with scaffold mesh size and modulus to a similar degree, levels of collagen and serum response factor (SRF), a key regulator of SMC phenotype, were more strongly correlated with mesh size. To gain insight into the cell signaling underlying these observed correlations, variations in cell metabolic state and in RhoA signaling were semi-quantitatively evaluated. Both RhoA activity, which is largely modulated by scaffold mechanics in 2D, and cell metabolic activity were highly correlated with hydrogel mesh size. These results indicate that the effects of scaffold mechanics on RhoA activity in 3D may be distinct from those in 2D and underscore the need for uncoupled investigation of scaffold parameters on cell behavior. Furthermore, the present data suggest that RhoA signaling and cell metabolic regulation may be closely linked.