Peripheral nerve injury (PNI) affects millions of individuals in the United States alone and can result in loss of motor and sensory function. The native repair response in the peripheral nervous system (PNS) enables peripheral nerve regeneration over short distances. The regenerative capacity of the PNS is often attributed to the immense plasticity of Schwann cells, the primary glia (neuronal support cell) present in the PNS. Following injury, mature Schwann cells can shift into a repair cell phenotype to better support regeneration. In cases of traumatic PNI, Schwann cells may fail to sustain this repair phenotype for the duration necessary and, thus, clinical intervention is required to enable complete regeneration and functional restoration. Synthetic biomaterial nerve grafts are readily investigated to bridge traumatic PNI gaps and improve regenerative outcomes. However, synthetic nerve grafts require further optimization to address the complex pathophysiology of the PNI environment and enable robust regeneration and function restoration. Electrospun fibers are often incorporated into synthetic nerve grafts to improve their regenerative capacity for preclinical studies. The diameter and orientation of electrospun fibers can be modified during the electrospinning process to produce a fibrous mat or graft filler that better mimics the native PNS extracellular matrix (ECM) to support and guide regenerating tissue. However, electrospun fibers alone are often unable to facilitate complete regeneration across critical length injury gaps. Loading the electrospun fibers with therapeutic molecules such as drugs, proteins, and nucleic acids that modulate the Schwann cell response and directly influence axonal regeneration can further improve the regenerative capacity of electrospun fibers. This thesis aims to develop aligned electrospun fiber-mediated drug and gene delivery platforms to improve the regenerative potential of the electrospun fibers for future use in synthetic peripheral nerve grafts.
First, we fabricated and characterized aligned electrospun poly(lactic-co-glycolic acid) (PLGA) fibers encapsulating the immunomodulatory drug fingolimod. Fingolimod holds the potential to target the complex pathophysiology of PNI by stimulating a repair Schwann cell phenotype and endogenous trophic factor production and neurite outgrowth from neurons. In vitro characterization revealed that the fingolimod-releasing electrospun fibers provided sustained release of fingolimod for 28 days, increased neurite outgrowth from whole dorsal root ganglia (DRG) explants and dissociated DRG neurons, increased Schwann cell migration outwards from the DRG body, and decreased Schwann cell mRNA expression levels of several myelin-associated factors. However, the fingolimod-releasing fibers did not increase Schwann cell mRNA expression levels of repair-associated, pro-regenerative factors. These findings indicate that the aligned fingolimod-releasing electrospun fibers likely released enough fingolimod to increase neurite outgrowth and Schwann cell migration but not enough to stimulate a repair Schwann cell phenotype. To our knowledge, this is the first electrospun fiber fingolimod delivery system designed for PNI applications.
Next, we aimed to further enhance the regenerative capacity of the aligned electrospun fibers for PNI applications by creating an aligned electrospun fiber platform for local delivery of mRNA encoding neurotrophin-3 (NT-3). NT-3 holds the potential to target the complex pathophysiology of PNI by supporting Schwann cell and neuron survival, stimulating Schwann cell migration, enhancing neurite outgrowth and axonal regeneration, and supporting remyelination of regenerated axons. Additionally, delivery of mRNA enables the transient expression and secretion of NT-3 protein local to the injury and bypasses several limitations presented by the delivery of proteins that possess low stability and, thus, a short half-life. First, we demonstrated the successful synthesis of bioactive mRNA encoding NT-3 and complexed the mRNA to a cationic transfection agent to form a cationic lipoplex. Next, we immobilized the mRNA lipoplexes to the surface of electrospun fibers with high efficiency through electrostatic interactions, enabling the sustained release of the mRNA lipoplexes over 28 days. In vitro characterization revealed that the aligned electrospun fiber-mediated mRNA delivery platform sustained increased levels of NT-3 protein secretion from Schwann cells for 21 days and stimulated increased neurite outgrowth from whole DRG explants. To our knowledge, this is the first electrospun fiber mRNA delivery system designed for tissue engineering applications. The findings detailed in this thesis provide a basis for further optimization of these electrospun fiber-mediated drug and gene delivery platforms for future testing in a preclinical PNI model.;
May 2022; School of Engineering
Dept. of Biomedical Engineering;
Rensselaer Polytechnic Institute, Troy, NY
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