Date of Award
PhD (Doctor of Philosophy)
Professor Fergal J. O'Brien
Irish Research Council
Peripheral Nerve Repair, Tissue Engineering, Gene Delivery, Neurotrophic Factors
Despite the success of tissue engineered nerve guidance conduits (NGCs) for the treatment of small peripheral nerve injuries, autografts remain the clinical gold standard for larger injuries. Therefore, there is a substantial unmet clinical need for the development of more advanced NGC-based therapies for peripheral nerve repair. In this context, the overall objective of the research presented in this thesis was to develop an advanced NGC-based therapeutic for peripheral nerve repair, initially by engineering a neuroconductive and physicochemically optimized biphasic NGC, which was then functionalized for the controlled delivery of neurotrophic factors in order to accelerate nerve repair. Finally, this biphasic NGC was utilized as an innovative non-viral gene delivery platform in order to elicit a sustained but transient therapeutic response from endogenous cells.
In Chapter 2, a biphasic NGC was successfully developed, combining a physicochemically optimized collagen-based outer conduit to bridge the site of injury, and a neuroconductive hyaluronic acid (HyA)-based luminal filler to support nerve repair across it. The natural polymers collagen and HyA were selected as the primary biomaterial components of the NGC – taking advantage of their biocompatibility, biodegradability, and functional properties, including collagen’s mechanical tunability, and HyA’s neuroconductivity. Altering the degree of crosslinking and collagen density allowed us to fine-tune a highly permeable and porous construct to limit the potential risk of neuroma formation, with physiologically relevant tensile strength and resistance to enzymatic degradation. The addition of laminin, an important basal lamina component, to the luminal filler, significantly enhanced the in vitro biological response supporting Schwann cell attachment, neural progenitor cell differentiation and axonal outgrowth from adult dorsal root ganglia. Ultimately, the in vivo efficacy of the biphasic NGC was demonstrated in terms of both morphological and functional recovery across a 10 mm sciatic nerve injury in rats.
While the results from Chapter 2 were promising, in Chapter 3 we sought to enhance the regenerative capacity of the biphasic NGC by utilizing poly(lactic-co-glycolic acid) (PLGA) microparticles in order to control the delivery of two neurotrophic factors, nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF). PLGA microparticles were found to be suitable carriers capable of controlling neurotrophic factor delivery with biphasic release kinetics, which were characterized by an immediate diffusion-mediated burst release followed by degradation-mediated release by day 14-21, complementing the expression of NGF and GDNF following injury. When the optimized microparticles were incorporated into the luminal filler component of the NGC, they were shown to exhibit long-term, tandem, and controlled release of NGF and GDNF with sustained bioactivity, which was capable of enhancing the biological activity of both neuronal cells and Schwann cells in a dose-dependent manner. When both neurotrophic factors were delivered in combination, they were found to further accelerate neurite outgrowth, Schwann cell neurotrophic factor production and migration, and axonal outgrowth from adult dorsal root ganglia.
In Chapter 4, a highly novel gene activated NGC for peripheral nerve repair was developed by incorporating non-viral polyethyleneimine-plasmid DNA (PEI-pDNA) nanoparticles, which were shown to be highly efficient for transfecting Schwann cells without significant associated cytotoxicity. When this system was used to deliver genes encoding for NGF, GDNF and the transcription factor c-Jun, it was shown to be capable of eliciting a positive therapeutic response from endogenous Schwann cells and neuronal cells. While all three genes showed therapeutic potential, delivery of the gene encoding for c-Jun showed the greatest capacity to accelerate axonal outgrowth and Schwann cell cytokine production. Ultimately, our gene activated NGC construct was shown to be capable of transfecting both Schwann cells and neuronal cells, exhibiting sustained but transient levels of enhanced neurotrophic factor protein production over time while demonstrating therapeutic potential by accelerating neurite outgrowth.
In summary, the research presented in this thesis has led to the development of a novel biphasic NGC for peripheral nerve repair, which is capable of being used as a platform for the controlled delivery of neurotrophic factor or as a gene activated system offering an innovative and cost-effective approach to control the timing, release and provide a sustained level of therapeutic protein production to potentially accelerate nerve repair.
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Lackington W. Advanced Scaffold-Based Therapeutics for Peripheral Nerve Repair [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2018.
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