Functionalization of electrospun poly(ε-caprolactone) fibers with the extracellular matrix-derived peptide GRGDS improves guidance of schwann cell migration and axonal growth.

The best available treatment of peripheral nerve lesions involves transplantation of an autologous nerve. This approach, however, entails sensory deficits at the donor site and requires additional surgery. Such limitations have motivated the search for a bioengineering solution to design artificial implants. For this purpose we are producing orientated biodegradable microfibers of poly(ε-caprolactone) (PCL) with electrospinning. The present study describes the functionalization of these electrospun fibers with biologically active peptides to produce guidance structures for Schwann cell migration and axonal regeneration. For the chemical modification PCL was blended with star-shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (PCL/sPEG) as a covalent linker for the peptide GRGDS, derived from extracellular matrix proteins. To test biological functions of electrospun fibers, Schwann cell migration and axonal growth from dorsal root ganglia explants were investigated with time lapse video microscopy. Migrating Schwann cells as well as growing sensory axons closely followed the electrospun fibers with occasional leaps between adjacent fibers. Cell migration was characterized by frequent changes in velocity and direction reversals. Comparison of substrates showed that functionalized fibers caused more Schwann cells to move out of the explants, supported faster cell migration and axonal growth than the nonfunctional fibers. Using inhibitors of intracellular signaling kinases, we found that these biological effects required activation of the phosphatidyl inositol-3-kinase pathway. Since sPEG-containing fibers also showed low levels of nonspecific protein adsorption, which is desirable in the context of artificial implant design, the peptide modification of fibers appears to provide good substrates for nerve repair.

Human neural cell interactions with orientated electrospun nanofibers in vitro.

AIM: Electrospun nanofibers represent potent guidance substrates for nervous tissue repair. Development of nanofiber-based scaffolds for CNS repair requires, as a first step, an understanding of appropriate neural cell type-substrate interactions. MATERIALS & METHODS: Astrocyte-nanofiber interactions (e.g., adhesion, proliferation, process extension and migration) were studied by comparing human neural progenitor-derived astrocytes (hNP-ACs) and a human astrocytoma cell line (U373) with aligned polycaprolactone (PCL) nanofibers or blended (25% type I collagen/75% PCL) nanofibers. Neuron-nanofiber interactions were assessed using a differentiated human neuroblastoma cell line (SH-SY5Y). RESULTS & DISCUSSION: U373 cells and hNP-AC showed similar process alignment and length when associated with PCL or Type I collagen/PCL nanofibers. Cell adhesion and migration by hNP-AC were clearly improved by functionalization of nanofiber surfaces with type I collagen. Functionalized nanofibers had no such effect on U373 cells. Another clear difference between the U373 cells and hNP-AC interactions with the nanofiber substrate was proliferation; the cell line demonstrating strong proliferation, whereas the hNP-AC line showed no proliferation on either type of nanofiber. Long axonal growth (up to 600 microm in length) of SH-SY5Y neurons followed the orientation of both types of nanofibers even though adhesion of the processes to the fibers was poor. CONCLUSION: The use of cell lines is of only limited predictive value when studying cell-substrate interactions but both morphology and alignment of human astrocytes were affected profoundly by nanofibers. Nanofiber surface functionalization with collagen significantly improved hNP-AC adhesion and migration. Alternative forms of functionalization may be required for optimal axon-nanofiber interactions.

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.