Structural Stiffness Gradient along a Single Nanofiber and Associated Single Cell Response
Abstract
Cell-substrate interactions are important to study for development of accurate in vitro research platforms. Recently it has been demonstrated that physical microenvironment of cells directly affects cellular motility and cytoskeletal arrangement. Specifically, previous studies have explored the role of material stiffness (Young\'s modulus: N/m2) on cell behavior including attachment, spreading, migration, cytoskeleton arrangement (stress fiber and focal adhesion distribution) and differentiation.
In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment. Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform. These fibers present a gradient of structural stiffness to the cells at constant material stiffness. Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites.
These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously. From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future.
In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment. Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform. These fibers present a gradient of structural stiffness to the cells at constant material stiffness. Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites.
These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously. From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future.
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