FIG. 3.

Real-time polymerase chain reaction (PCR) analyses of various vSMC differentiation-related markers when cultured on the different substrata. Interestingly, for the intermediate filament-related markers calponin and desmin and for the smooth muscle-specific cytoskeletal protein smoothelin, soft ANFS resulted in statistically higher gene expression. This highlights the synergetic effects of substratum nanotopography and elasticity on vSMC differentiation, with ANFS resulting in a more profound effect on vSMC-restricted contractile protein marker genes. Interestingly, for the less stringent marker smooth muscle α-actin (α-SMA) (also found in myofibroblasts), there was no statistical difference between soft and stiff ANFS. For tropomyosin, found to be erratically upregulated in hypertension, stiff ANFS expressed statistically higher levels. Statistically significant difference between nanopatterned and unpatterned substrata (p<0.05) for the same substratum stiffness is indicated with an asterisk. For all other substrata-type combinations, the identical signage indicates statistically significant difference for that pair.