Optimization of fully aligned bioactive electrospun fibers for in vitro nerve guidance

Complex architecture of natural tissues such as nerves requires the use of multifunctional scaffolds with peculiar topological and biochemical signals able to address cell behavior towards specific events at the cellular (microscale) and macromolecular (nanoscale) level. In this context, the electrospinning technique is useful to generate fiber assemblies having peculiar fiber diameters at the nanoscale and patterned by unidirectional ways, to facilitate neurite extension via contact guidance. Following a bio-mimetic approach, fully aligned polycaprolactone fibers blended with gelatin macromolecules have been fabricated as potential bioactive substrate for nerve regeneration. Morphological and topographic aspects of electrospun fibers assessed by SEM/AFM microscopy supported by image analyses elaboration allow estimating an increase of fully aligned fibers from 5 to 39 % as collector rotating rate increases from 1,000 to 3,000 rpm. We verify that fully alignment of fibers positively influences in vitro response of hMSC and PC-12 cells in neurogenic way. Immunostaining images show that the presence of topological defects, i.e., kinks-due to more frequent fiber crossing-in the case of randomly organized fiber assembly concurs to interfere with proper neurite outgrowth. On the contrary, fully aligned fibers without kinks offer a more efficient contact guidance to direct the orientation of nerve cells along the fibers respect to randomly organized ones, promoting a high elongation of neurites at 7 days and the formation of bipolar extensions. So, this confirms that the topological cue of fully alignment of fibers elicits a favorable environment for nerve regeneration.