Deposition of electrospun fibers on reactive substrates for in vitro investigations.

Recent in vitro studies with electrospun nanofibers have used a range of techniques. The in vitro system presented in this article describes electrospun fibers deposited onto chemically reactive substrates to provide fiber adherence and surface chemistry control of the substrate. Fibers of poly(epsilon-caprolactone) (PCL) or of a blend of PCL and collagen type I (C/PCL) were electrospun directly onto collectors coated with isocyanate-terminated star (polyethylene glycol) (sPEG). Alternatively, parallel electrospun fibers were collected on dual collectors in "dilute" quantities and transferred onto sPEG-coated substrates. The initial reactive nature of the substrates allows the collection of very few fibers, which adhere well during frequent washes. Furthermore, the sPEG layer transforms into protein-repellent substrates with the additional potential to include specific cellular mediators such as glycine-arginine-glycine-aspartate-serine (GRGDS) peptides to promote cell adhesion. Therefore, the fiber and substrate chemistry can be modified independently, which is particularly useful for in vitro studies of guided migrating cells. In the present work, dissociated cells of dorsal root ganglia seeded onto the substrates were investigated to assess the influence of different combinations of fiber material, fiber orientation, and surface functionalization. Cell adhesion was observed predominantly on the nanofibers, except when the sPEG layer on the substrate contained GRGDS. On the cell-repellent sPEG substrates, neurites were aligned in direct contact with parallel C/PCL fibers and less so with PCL fibers. In contrast, neurite alignment showed less guidance effect with C/PCL electrospun fibers on the GRGDS/sPEG-coated substrates. Therefore, the combination of oriented biologically active fibers on cell-repellent surfaces enhanced the guidance of such cells. These reactive substrate systems provide a multitude of in vitro combinations for providing cells with specific mediators and, in turn, defining the optimum environment of regenerating devices for in vivo studies.