The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

Abstract

Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(L-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.

Electrospun Nanofibrous Materials for Neural Tissue Engineering

“…Mouse embryonic stem cell differentiation into neurons, astrocytes, and oligodendrocytes was enhanced when cultured on aligned and random PCL fibers [19]. In other studies, aligned PLLA nanofibrous scaffold also supported the growth and extension of dorsal root ganglion (DRGs) [20,21] and primary motor neurons [20]. Primary motor neurons developed neurites earlier on PLLA nanofibers when compared to PLLA films [22].…”

Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

“…Fig. 1.7 ES has been demonstrated to be an extremely versatile technology able to produce fibres with diameters ranging from few nanometers to several microns, from over two hundred synthetic and natural polymers, in the form of plain fibres, blends and organic-inorganic composite fibres [148][149][150]. Another advantage is the simplicity of the process that does not require any sophisticated and expensive equipment and that can be easily scaled-up for mass production.…”

“…In order to circumvent the above discussed drawbacks, information similar to that provided by immunoistochemistry can be obtained by immunofluorescence performed directly on nanofibrous scaffolds [138,140,141,148]. In this case the antibody is tagged with a fluorophore which is visualized under the UV-light microscope.…”

Novel opportunities and challenges offered by nanobiomaterials in tissue engineering

“…In vitro cell culture experiments have been carried out with fi bers of biocompatible and biodegradable polymers of natural and synthetic origin such as poly(glycolic acid) (PGA), poly(l-lactic acid) (PLLA), poly(ε-caprolactone) (PCL), as well as copolymers from the corresponding monomers in various compositions, segmented polyurethanes, polyphosphazenes, gelatin, collagen, and chitosan (Chen et al 2008;Choi et al 2008;Corey et al 2008;Powell and Boyce 2008). A broad spectrum of cells has been seeded on electrospun scaffolds: for instance keratinocytes, chondrocytes, osteoblasts, mesenchymal stem cells, Schwann cells, neural stem cells, smooth muscle cells, osteoblasts, fi broblasts, and endothelial cells.…”