Tissue engineering holds enormous potential to revolutionize the field of medicine in general, and cardiology specifically, by providing replacement tissues for the human body. In order to engineer functional tissue replacements, it is increasingly clear that a variety of inputs must be provided to cells with precise spatial and temporal control to direct tissue development. These inputs include not only soluble (e.g., growth factors, mitogens, etc.) and insoluble (e.g., extracellular matrix (ECM)) biochemical cues, but also mechanical cues to dictate cell fate. This is particularly true for vascular smooth muscle, a tissue that is normally subjected to cyclic mechanical strain in vivo and that can experience significant changes in passive mechanical properties as a result of numerous pathologies.

Motivated by the need for a suitable model system in which to analyze the coordinated effects of ECM chemistry and mechanics on smooth muscle cell (SMC) phenotype, we have adapted poly(ethylene glycol) (PEG)-based synthetic ECM analogs due in part to the fact that their mechanical and cell adhesive properties can be independently controlled. As expected, we found that SMC attachment and spreading occur on RGD and full length ECM-modified PEG surfaces, but not on unmodified control surfaces or on surfaces with a scrambled peptide sequence (GRDGS) after 24 hours of incubation, regardless of serum content in the media. Interestingly, for a given ligand density, the extent of 2-D SMC spreading was not significantly influenced by ECM mechanics across a wide range of elastic moduli values (13.7 kPa – 423.9 kPa). Likewise, for a given elastic modulus, SMC spreading was not significantly influenced by RGD, KQAGDV (a fibrinogen-derived peptide sequence), fibronectin, or type I collagen density across a wide range of ligand densities. On the other hand, quantitative analysis of focal adhesions in SMCs cultured on these surfaces revealed that both their size and the elongation were highly dependant on the substrate stiffness as well as the ligand type and density coupled to PEG.

We also analyzed the proliferation and differentiation of SMCs as a function of PEG stiffness on the surface of RGD-modified substrates. After 7 days of culture, SMCs proliferated 3.9-fold on the stiffest substrate tested (423.9 kPa) yet only 2.4-fold on the softest substrate (13.7 kPa). In exciting contrast, we found that the extent of SMC differentiation increased with PEG compliance, where the association of the SMC-specific differentiation markers calponin and caldesmon with α-actin fibrils was highest on the softest substrates tested. This suggests that the shift of SMCs from a synthetic (proliferative) to a contractile (differentiated) phenotype can be manipulated by controlling the substrate's mechanical properties. Currently, we are translating our studies of SMC-material interactions into a more realistic 3-D model, and will present data demonstrating the utility of PEG-based materials as ideal in vitro models to address how ECM mechanics, ligand identity, and ligand density cooperatively regulate the phenotypic conversion of SMCs from a synthetic to a contractile phenotype in a 3-D model system as well.