Bhangra, Kulraj Singh; (2020) Delivery and integration of Engineered Neural Tissue for peripheral nerve repair. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Peripheral nerve injury is a debilitating disorder affecting approximately one million people in Europe and US annually. The clinical gold standard treatment for repairing a long-gap nerve defect remains the nerve autograft, albeit functional recovery is inadequate. In addition, disadvantages include donor site morbidity, limited availability and multiple surgeries suggesting there is a clear need to develop effective alternatives that mimic key autograft features. To directly address this clinical need and to mitigate the disadvantages of the autograft Engineered Neural Tissue (EngNT) has been developed for repairing long-gap nerve defects. EngNT is a robust collagen hydrogel formed from columns of aligned therapeutic cells that can direct and promote neurite outgrowth in vitro and in vivo. Previous work has shown that EngNT can be developed with different stem cells and more recently blends of biomaterials have also been investigated. Nevertheless, there are still challenges associated with the delivery and integration of EngNT. In particular, previous work has noted limited neuronal ingrowth into EngNT seeded with SCL4.1/F7 Schwann cells. This thesis incorporates stiff bioresorbable phosphate glass fibres (PGFs) with EngNT to bridge the interface between a damaged proximal nerve stump and EngNT. Results show that PGFs can be optimised through altering their diameter and coating their surface with laminin and fibronectin to promote robust orientated neuronal growth across the proximal nerve stump - EngNT interface, in vivo. Furthermore, when EngNT was seeded with differentiated human dental pulp stem cells (d-hDPSCs) to repair a critical gap defect, results showed limited neuronal regeneration but increased number of blood vessels. This effect was hypothesised to be attributable to low oxygen levels within the construct and tested within this thesis. Modest changes in VEGF-A, NGF and BDNF expression were detected when collagen constructs seeded with d-hDPSCs were exposed to low oxygen conditions in vitro. In addition, secretome from d-hDPSCs exposed to 3% oxygen increased endothelial cell proliferation but reduced neurite length in comparison to secretome from d-hDPSC cultured at 16% oxygen. These results are consistent with the original hypothesis and highlight the importance of local oxygen levels in determining the efficacy of engineered tissues. Previously, commercially available NGC materials have been used to deliver EngNT into a critical gap defect. However, these tubes are predominately designed to have patent lumens and are inherently stiff. This study includes a detailed mechanical characterisation of the bulk and dynamic viscoelastic properties of nerve tissue and prospective nerve guidance conduit materials for the delivery of EngNT. Lastly, this thesis investigates different serum-free cryoprotectants for the long-term storage of EngNT to ensure it is readily available as an off-the-shelf product. Results show that the use of poly (propylene glycol) with 7.5% DMSO is a suitable option that retains cell viability. Future work involves the continued multidisciplinary effort of developing EngNT with the aid of mathematical models to streamline the design process. Data generated from experiments such as those described above will be part of a large data bank that feeds into and parameterises the mathematical models to allow them to accurately simulate and test different EngNT designs for peripheral nerve repair.

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