Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications

Abstract

Aligned, electrospun polymer fibers have shown considerable promise in directing regenerating axons in vitro and in vivo. However, in several studies, final electrospinning parameters are presented for producing aligned fiber scaffolds, and alignment where minimal fiber crossing occurs is not achieved. Highly aligned species are necessary for neural tissue engineering applications to ensure that axonal extension occurs through a regenerating environment efficiently. Axonal outgrowth on fibers that deviate from the natural axis of growth may delay axonal extension from one end of a scaffold to the other. Therefore, producing aligned fiber scaffolds with little fiber crossing is essential. In this study, the contributions of four electrospinning parameters (collection disk rotation speed, needle size, needle tip shape and syringe pump flow rate) were investigated thoroughly with the goal of finding parameters to obtain highly aligned electrospun fibers made from poly-L-lactic acid (PLLA). Using an 8 wt% PLLA solution in chloroform, a collection disk rotation speed of 1000 revolutions per minute (rpm), a 22 gauge, sharp-tip needle and a syringe pump rate of 2 ml h(-1) produced highly aligned fiber (1.2-1.6 microm in diameter) scaffolds verified using a fast Fourier transform and a fiber alignment quantification technique. Additionally, the application of an insulating sheath around the needle tip improved the rate of fiber deposition (electrospinning efficiency). Optimized scaffolds were then evaluated in vitro using embryonic stage nine (E9) chick dorsal root ganglia (DRGs) and rat Schwann cells (SCs). To demonstrate the importance of creating highly aligned scaffolds to direct neurite outgrowth, scaffolds were created that contained crossing fibers. Neurites on these scaffolds were directed down the axis of the aligned fibers, but neurites also grew along the crossed fibers. At times, these crossed fibers even stopped further axonal extension. Highly aligned PLLA fibers generated under optimized electrospinning conditions guided neurite and SC growth along the aligned fibers. Schwann cells demonstrated the bipolar phenotype seen along the fibers. Using a novel technique to determine fiber density, an increase in fiber density correlated to an increase in the number of neurites, but average neurite length was not statistically different between the two different fiber densities. Together, this work presents methods by which to produce highly aligned fiber scaffolds efficiently and techniques for assessing neurite outgrowth on different fiber scaffolds, while suggesting that crossing fibers may be detrimental in fostering efficient, directed axonal outgrowth.

Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity

“…PLLA filaments have been proposed as a promising tool for axon growth and guidance [67]. A special feature of our conduit is a triple-layer porous structure, which results in a more specific pore architecture than comparable solutions [68].…”

Aligned electrospun polymer fibres for skeletal muscle regeneration

“…Previous studies have shown that aligned fibres can influence the orientation of the cell type seeded upon them (Bashur et al, 2006;Bian and Bursac, 2009;Choi et al, 2008;Courtney et al, 2006;Huang et al, 2006;Lehnert et al, 2004;Riboldi et al, 2005;Wang et al, 2009). Other studies have incorporated natural biologic materials e.g.…”

“…Alignment of polymer nanofibres can be controlled by selecting the relevant collector plate: stationary or very slow rotation of the collector plate can be used to manufacture randomly oriented fibres, whereas a highspeed rotating mandrel can be used to create aligned fibres (Bian and Bursac, 2009;Blackwood et al, 2008;Courtney et al, 2006;Wang et al, 2009).…”

Electrospun Nanofibrous Materials for Neural Tissue Engineering

“…This allows for the fiber to collect along the direction of rotation. Small diameter tubes can also be fabricated by this method and have been used in vascular repair studies [16]. A high rotation speed produces increased fiber alignment as compared to lower rotation speed, but may cause fiber discontinuity [15,17].…”