Biomechanistic Study of Smooth Muscle Cell Sheet during Circumferential Alignment in Circular Micropatterns

Engineered functional blood vessels play an important role in tissue regeneration and wound healing, but they remain far from clinical practices because of the complex three-layer capillary histology. In general, the reconstruction of tunica media from vascular smooth muscle cells (SMCs) that mediate vasoconstriction and vasodilation of circulatory system is critical for engineering functional blood vessels. To date, the mechanotransduction during circumferential alignment of SMC layer in tunica media remains to be elucidated. In this study, concentric microwalls fabricated on a polydimethylsiloxane (PDMS) substrate were developed for cell traction force microscopy (CTFM) of SMC layer. First, the cell shape, density, and alignment orientation of SMCs during circumferential cell sheet formation were measured during 7-day cell culture. Second, a collective CTFM assay was carried out to measure the traction forces exerted by the confluent cell layer at various stages of circumferential alignment. It was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Moreover, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. The immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR) analyses of actin filaments further revealed the relation between the monolayer-generated traction forces and the direction of aligned actin filaments.