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

Abstract

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.

Regenerative nanomedicine: current perspectives and future directions

“…58 An excellent review by Biazar et al discusses various types of biologic nerve conduits and synthetic nerve guides. 59 Cirillo et al 60 …”

“…These fully aligned nanofibers act as a topological cue for neurite growth, thereby improving nerve regeneration. 60 The in vivo studies of intraluminal nanofibrous nerve guides for nerve injury repair have shown considerable improvement in nerve growth potential. The conduits are coated with laminin and nerve growth factors for this purpose.…”

Highly polydisperse keratin rich nanofibers: Scaffold design and in vitro characterization

“…The use of fibrous proteins allows reproducing during the regeneration processes the pattern of chemical and morphological cues typically exerted by natural collagen in vivo at the site of injury. 9,10 In this context, alternative proteins have been more recently tested alone or in combination with other polymers to design protein-based fibrous scaffolds with peculiar functionalities for tissue engineering applications. In particular, the use of collagen for the fabrication of nanofibers via electrospinning technique has been long time considered an effective strategy to design fiber scaffolds with different, morphology, structural arrangement, surface roughness, and porosity, by an accurate control of process parameters and solution properties.…”

“…In order to overcome these limitations, collagen or its denatured protein-that is, gelatin-have been used in the crosslinking form, 2 or in combination with biodegradable polymers including poly(lactic acid), 3,4 poly(glycolic acid) 5 and their copolymer poly(lactic-co-glycolic acid) (PLGA), 6 and poly(ε-caprolactone) (PCL) 7,8 to improve the in vitro and in vivo biomechanical stability. 9,10 In this context, alternative proteins have been more recently tested alone or in combination with other polymers to design protein-based fibrous scaffolds with peculiar functionalities for tissue engineering applications. For instance, silk-based materials have been largely considered for their capability to improve proliferation also supporting mechanical properties to the scaffold.…”

Atomic Force Microscopy: A Powerful Tool to Address Scaffold Design in Tissue Engineering

“…AFM morphological images of ( A ) solvent casting poly(ε-caprolactone) (PCL) films (scan area 50 × 50 µm 2 ) [53]: ( B ) PCL-Gel electrospun membrane (scan area 20 × 20 µm 2 ) [54]; ( C ) PLGA electrosprayed microparticle (scan area 20 × 20 µm 2 ).…”