Busuttil, Francesca; (2019) Combining tissue engineering and gene delivery to enhance peripheral nerve regeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There has been an increased interest in the development of nerve repair devices to improve peripheral nerve regeneration following injury. The aim of this study was to investigate whether nerve repair devices containing genetically modified cells overexpressing VEGF-A165 would augment regeneration. An in vitro proof-of-concept study was carried out to deliver marker genes (luciferase and eGFP) to a rat Schwann cell line (SCL4.1/F7) using a lentiviral vector. The transduced cells were used to produce engineered neural tissue and bioluminescence imaging was used to assess cell viability in the constructs. It was initially thought the presence of the luciferase gene in the expression cassette would allow real-time and sustained imaging of the cells in the engineered neural tissue. However, while bioluminescence imaging provided an indication of cell viability in vitro, it proved to be ineffective for in vivo and ex vivo imaging. Having established that SCL4.1/F7 cells were amenable to lentiviral transduction, a lentiviral vector delivering the VEGF-A165 gene was designed and produced. The VEGF-A165 produced by the transduced SCL4.1/F7 cells increased endothelial cell viability, migration and tube formation in vitro. It also increased SCL4.1/F7 cell proliferation and migration. SCL4.1/F7 cells overexpressing VEGF-A165 were found to increase endothelial cell network formation and neurite length in 3D co- culture models. Foetal human neural stem cells and rat adipose derived stem cells were also successfully transduced with the lentiviral vector delivering VEGF-A165. Based on the in vitro results, it was postulated that EngNT made from SCL4.1/F7 cells overexpressing VEGF-A165 implanted into a rat model of sciatic nerve injury would enhance regeneration. Unexpectedly, a pilot study revealed that this did not result in improved functional recovery or increased axon and blood vessel counts compared to controls. The results from this study highlight that attention needs to be paid to the dose and duration of expression of VEGF-A165 to optimise both its angiogenic and neurotrophic effects.

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