Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.759833
Title: Material selection for fabricating an internal guidance scaffold for improving current nerve guide conduits
Author: Taylor, Caroline
ISNI:       0000 0004 7431 8521
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Abstract:
Peripheral nerve injuries affect 2.8% of all trauma patients which amounts to 8.5 million 'restricted activity days' every year. Unlike the central nervous system, nerves of the peripheral nervous system can regenerate after injury, at a rate of around 1 mm/day. Current treatments involve direct end to end suturing or using an autograft, which is the current 'gold standard' treatment to bridge large gaps (over 20mm). However, there is limited donor nerve available and limited nerve function is restored. Current nerve guide conduits (NGCs) are fabricated from both natural and synthetic materials. However, the efficiency of current nerve guide conduits is limited and conduits only treat defects up to 20mm. Research focuses primarily on improving hollow nerve guide conduits in several different ways. These include: the addition of intraluminal guidance scaffolds, coatings to modify surface chemistry or changing the conduit design by incorporating channels and pores. The aim of this work was to investigate different blends of synthetic polymers for their potential use as an intraluminal nerve guidance scaffold. It is hypothesised that the addition of electrospun fibres to hollow nerve guide conduits will increase nerve regeneration length by providing aligned guidance for Schwann cell attachment, migration and nerve regeneration. Blends of synthetic polymers were characterised to assess surface properties, mechanical properties, biocompatibility and cytotoxicity. Electrospinning was performed to determine if polymers, and their blends, could be fabricated into aligned fibres of varying diameters. Polymers which had a higher Young's modulus, Polyhydroxybutyrate-co-Valerate (PHBV), Polycaprolactone (PCL), Polyhydroxybutyrate (P(3HB) and Polylactic Acid (PLLA) could all be fabricated into aligned fibres of varying diameters. Polymers with more elastomeric properties Polyhydroxyoctanoate (P(3HO) and Polyhydroxyoctanoate-cohydroxydecanoate (P(3HO-co-3HD) could not be electrospun into aligned fibres. Overall, the most efficient blend ratio of the brittle polymers with the elastomeric polymers was 50:50. This ratio fabricated aligned fibres and maintained a lower Young's modulus (closer to that of native nerve). The effect of fibre diameter on neuronal and Schwann cell adherence, viability and differentiation was investigated by culturing cells on electrospun Polyhydroxybutyrate-coValerate (PHBV) fibres. In vitro analysis concluded that 5 and 8μm diameter fibres supported neuronal cell viability and differentiation most effectively. The effect of different material composition on neuronal and Schwann cell adherence, viability and differentiation was investigated by culturing cells onto 5 and 8 μm fibres of PHBV, PCL, P(3HB): P(3HO) (50:50) and P(3HO-co-3HD):PLLA (50:50). PCL was investigated as a control material. PHBV, P(3HB):P(3HO) (50:50) and P(3HO-co-3HD):PLLA (50:50) were investigated further due to success with regards to electrospinning, excellent cell viability and mechanical properties compared to other investigated blends of polymers. It was hypothesised that using polyhydroxyalkanoates, and novel blends of these polymers, would improve neuronal and Schwann cell attachment and support cell proliferation and differentiation more effectively than known FDA approved biopolymers, due to their chemical composition. The novelty in this work is the use of PHA blends and the ability to electrospin them. NG108-15 neuronal cell neurite outgrowth was significantly higher when cells were cultured on the 8μm PHBV fibres compared to the other materials and fibre diameters. Schwann cell viability was significantly decreased when cultured on the 5 and 8μm P(3HOco-3HD):PLLA (50:50) fibres, compared to the other polymer blends. Polymer fibres were further investigated using a novel 3D ex vivo fibre testing model. 5mm Polyethylene glycol hollow tubes were manufactured using microstereolithography. Polymer fibres, of 5 and 8μm diameters, were threaded into tubes, and an explanted DRG body placed on the proximal end. The use of DRGs in neuronal cell culture mimics a nerve injury, as neurite extensions grow from the DRG body after removing the nerve roots. It was hypothesised that the addition of electrospun fibres to hollow nerve guide conduits would increase nerve regeneration length. Both Schwann cell migration lengths and neurite outgrowth lengths were greater on the 5μm fibres, compared to their 8μm counterparts. Schwann cell migration length was statistically greater on the 5μm PHBV fibres and P(3HO):P(3HB) (50:50) fibres. PHBV 5μm fibres supported greater neurite lengths, compared to the 8μm PCL and 8μm PHBV fibres. This study highlights the difference between using a neuronal cell line, compared to using a primary neuron (DRG) model, and using a 3D in vitro culture model compared to 2D in vitro culture. This work also reveals the potential of 5μm P(3HO):P(3HB) (50:50) and PHBV fibres, as intraluminal fibrous scaffolds to improve nerve repair and would support the use of this blend for in vivo investigations. Additionally, surface modification is used to improve current nerve guide conduits, by improving biocompatibility of the surfaces of bulk biodegradable polymers with the addition of reactive chemical groups. Compared to plasma polymerisation, aminosilanisation is an alternative method to modify surfaces with amine groups as it is cost effective, scalable and cheaper. Two different chain lengths, long and short, of aminosilanes were investigated, to determine their potential use in peripheral nerve repair. It was hypothesised that the addition of aminosilanes plain glass would improve neuronal and Schwann cell adherence and neuronal cell differentiation. The novelty of this work is that aminosilanes have not been investigated for use in peripheral nerve repair and that they could be a useful addition to biopolymer NGCs. Long chain aminosilanes supported significantly longer neurite lengths from NG108-15 neuronal cells, compared to the short chain and plain glass control. Both chain lengths supported NG108-15 neuronal and Schwann cell viability. A co-culture of primary neurons and Schwann cells was established by dissociating rat DRGs. Long chain aminosilanes supported significantly longer neurite lengths from primary neurons, compared to the short chain aminosilane, plain glass and tissue culture plastic controls. This work highlights the potential use of long chain aminosilanes for use in peripheral nerve repair, to coat conduits or intraluminal guidance scaffolds. Besides electrospinning, pressurized gyration has recently been reported to manufacture fibrous scaffolds for tissue engineering applications. The set up established at The Department of Mechanical Engineering, University College London, was used to manufacture P(3HB) and P(3HB):P(3HO-co-3HD) (80:20) fibres. Gyrated fibres were investigated with electrospun P(3HB) fibres, to determine which manufacturing method was the most efficient in manufacturing aligned fibres, and in vitro and ex vivo analysis was performed. SEM analysis confirmed that the P(3HB) electrospun fibres were more aligned compared to both gyrated fibre types. Both gyrated and electrospun fibres supported NG108-15 neuronal cell and Primary Schwann cell proliferation and viability. However, greater neurite lengths were observed on the electrospun P(3HB) fibres compared to the gyrated PHA fibres, when culturing DRGs. Schwann cell migration length was also significantly larger on the electrospun fibres compared to the gyrated fibres. This work highlights the potential of gyration for fabricating fibrous scaffolds. However, future work will need to be performed to fabricate aligned fibre scaffolds which compare with electrospun fibres. Overall, this thesis demonstrates that Polyhydroxyalkanoates, and blends of PHAs, can be fabricated into aligned fibre scaffolds by electrospinning. It also demonstrates that the addition of PHAs to polymer blends supports neuronal and Schwann cell attachment, proliferation and differentiation more effectively than the biopolymer alone. Although pressurized gyration is a new technique to fabricate fibre scaffolds, this thesis has shown that it does not fabricate fibres with the required alignment for nerve repair and therefore, more optimisation of the technique is required. Surface modification, using silanisation, has also been shown to be an effective technique to modify a glass surface with amine reactive groups, and therefore, can be applied to polymer surface modification.
Supervisor: Haycock, John W. ; Claeyssens, Frederik Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.759833  DOI: Not available
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