Development of an implantable multilayer three-dimensional scaffold consisting of aligned electrospun fibers for neuronal regeneration applications

There are 11,000 new spinal cord injuries (SCI) reported every year in the United States. After injury to the central nervous system (CNS), neurons do not grow through the injury site because of the presence of a glial scar that contains various inhibitory cues. Currently, there are no effective treatments to improve regenerative outcomes following CNS damage. Compared to the CNS injury, damage to the peripheral nervous system (PNS) is considerably more common. Approximately 300,000 individuals sustain injury to the PNS every year in the U.S. Nerve autografts and synthetic alternatives are currently used to repair large gaps following peripheral nerve injury. Even with these treatments, outcomes following nerve repair remain poor, resulting in full or partial paralysis. Therefore, it is imperative to develop an implantable three dimensional conduit to guide axons through the injury site.Electrospinning is a simple and versatile technique that produces ultrafine fibers made from various polymers. In this study, the optimum conditions for electrospinning aligned poly-L-lactic acid (PLLA) fibers were determined. The rotation speed of the collection disk, the size and shape of the needle tip were varied. The rate of fiber deposition (electrospinning efficiency) was improved by wrapping an insulator around the needle tip and the effect of the syringe pump flow rate was investigated. The fiber alignment and density were analyzed. It was determined that highly aligned fibers were created when the rotation speed of a collection disk was 1000 revolutions per minute (rpm), using a 22 gauge (GA) sharp needle, and a syringe pump flow rate of 2 ml/hr. The effects of fiber density on axonal outgrowth were also evaluated in vitro using embryonic stage nine chick dorsal root ganglia (E9 chick DRGs). The cell culture showed that the aligned PLLA fibers manufactured from optimized electrospinning conditions guided axonal outgrowth along the fiber. The density of axonal outgrowth was proportional to the PLLA fiber density.Based on these experimental results, an implantable multilayered three dimensional (3D) conduit was developed for neuronal regeneration applications. The conduit consisted of a double layer of PLLA films containing aligned, electrospun PLLA fibers. Fiber alignment on the film prior to conduit fabrication was characterized. Additionally, fiber morphology within the conduit was studied following conduit fabrication. Rolling the film into a three dimensional structure did not substantially affect fiber alignment. E9 chick DRGs were also cultured on a PLLA film (2D film), a PLLA film containing aligned, electrospun PLLA fibers (2D fibers), and within a conduit that contained aligned, electrospun PLLA fibers (3D fibers). Axonal outgrowth on 2D aligned fibers and within a 3D conduit was directed along the fibers, while axonal outgrowth on just the film did not grow in a directed manner. The results suggest that this multilayered 3D conduit containing aligned, electrospun fibers is a promising implantable scaffold that may organize and guide neuronal regeneration after CNS or PNS injury.