Novel fibrous scaffolds with dual growth factor delivery and non-viral gene delivery for neural tissue engineering

Abstract

Electrospun fibrous scaffolds capable of providing growth factor delivery and contact guidance have distinctive advantages for tissue engineering. Gene delivery may also be employed for tissue regeneration. This project aimed to investigate the formation, structure and characteristics/properties of bicomponent nanofibrous scaffolds for the dual delivery of glial cell line-derived growth factor (GDNF) and nerve growth factor (NGF), aligned-fiber scaffolds providing both biochemical and topographical cues for peripheral nerve tissue regeneration, new polymeric vectors for gene delivery, and composite scaffolds with the combination of nanofibrous scaffolds and gene delivery.

GDNF and NGF were incorporated into core-shell structured poly(lactic-co-glycolic acid) (PLGA) and poly (D,L-lactic acid) (PDLLA) nanofibers, respectively, through emulsion electrospinning. Using dual-source dual-power electrospinning (DSDP-ES), bicomponent scaffolds composed of GDNF/PLGA fibers and NGF/PDLLA fibers with different fiber component ratios were produced. The structure and properties, including in vitro release behavior and in vitro degradation, of mono- and bicomponent scaffolds were systematically investigated. Aligned-fiber bicomponent scaffolds were subsequently made using DSDP-ES and high-speed electrospinning. Nerve guidance conduits (NGCs) were formed using aligned-fiber scaffolds. To modulate the release behavior of GDNF and NGF, multi-layered fibrous scaffolds were fabricated through DSDP-ES and sequential electrospinning. Concurrent and sustained release of GDNF and NGF from bicomponent scaffolds was achieved and their release profiles could be tuned. The aligned-fiber topography and dual and sustained delivery of growth factors were realized in both aligned-fiber bicomponent scaffolds and NGCs. Spatio-temporal release of GDNF and NGF was achieved using multi-layered fibrous scaffolds.

For gene delivery, PLGA and poly(ethylenimine) (PEI)-based nanocomplexes (NCs) were fabricated using different methods. To improve the stability and transfection efficiency of NCs, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA-PEI-PEG-FA and PLGA-PEI-PEG-RGD copolymers were synthesized. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. Composite scaffolds composed of bicomponent fibrous scaffolds and PLGA-PEI-PEG-RGD/DNA NCs were eventually formed using a wetting and freeze-drying method.

In vitro biological investigations were conducted for scaffolds and NCs. The bioactivity of GDNF and NGF was preserved. Rat pheochromocytoma cells (PC12 cells) were found to attach, spread and proliferate on all scaffolds. The release of growth factors from scaffolds could induce much improved neurite outgrowth and neural differentiation. GDNF and NGF released from GDNF/PLGA scaffolds and NGF/PDLLA scaffolds, respectively, could induce dose-dependent neural differentiation separately. A synergistic effect of GDNF and NGF released from bicomponent scaffolds on promoting neural differentiation was found. The delivery of growth factors and aligned-fiber morphology induced neurite alignment and much enhanced neural differentiation in a synergetic manner. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios.