Stiffness of Aligned Fibers Regulates the Phenotypic Expression of Vascular Smooth Muscle Cells

Electrospun uniaxially aligned ultrafine fibers show great promise in constructing vascular grafts mimicking the anisotropic architecture of native blood vessels. However, understanding how the stiffness of aligned fibers would impose influences on the functionality of vascular cells has yet to be explored. The present study aimed to explore the stiffness effects of electrospun aligned fibrous substrates (AFSs) on phenotypic modulation in vascular smooth muscle cells (SMCs). A stable jet coaxial electrospinning (SJCES) method was employed to generate highly aligned ultrafine fibers of poly(L-lactide-co-caprolactone)/poly(L-lactic acid) (PLCL/PLLA) in shell-core configuration with a remarkably varying stiffness region from 0.09 to 13.18 N/mm. We found that increasing AFS stiffness had no significant influence on the cellular shape and orientation along the fiber direction with the cultured human umbilical artery SMCs (huaSMCs) but inhibited the cell adhesion rate, promoted cell proliferation and migration, and especially enhanced the F-actin fiber assembly in the huaSMCs. Notably, higher fiber stiffness resulted in significant downregulation of contractile markers like alpha-smooth muscle actin (alpha-SMA), smooth muscle myosin heavy chain, calponin, and desmin, whereas upregulated the gene expression of pathosis-associated osteopontin (OPN) in the huaSMCs. These results allude to the phenotype of huaSMCs on stiffer AFSs being miserably modulated into a proliferative and pathological state. Consequently, it adversely affected the proliferation and migration behavior of human umbilical vein endothelial cells as well. Moreover, stiffer AFSs also revealed to incur significant upregulation of inflammatory gene expression, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1), in the huaSMCs. This study stresses that although electrospun aligned fibers are capable of modulating native-like oriented cell morphology and even desired phenotype realization or transition, they might not always direct cells into correct functionality. The integrated fiber stiffness underlying is thereby a critical parameter to consider in engineering structurally anisotropic tissue-engineered vascular grafts to ultimately achieve long-term patency.