Improving peripheral nerve regeneration: a multimodal approach

Docherty, Robert Julian (2019) Improving peripheral nerve regeneration: a multimodal approach. PhD thesis, University of Glasgow.

Abstract

Injury to the peripheral nerve system (PNS) is a relatively common occurrence and can have a devastating impact on quality of life. Currently, autologous nerve graft remains the gold standard for clinical nerve injury repair, although this approach leads to associated donor site morbidity. Alternatives are the use of nerve guidance conduits (NGC), which have shown some success over small defects. Research to improve NGC require a combined approach of increasing and applying the understanding of the underlying neuronal behaviour and signalling mechanisms, as well as scaffolding materials that are conducive to neuronal survival, axon outgrowth and guidance. Here both of these aspects are addressed separately to provide the basis for a future improvement to regenerative strategies for PNS repair.

The combination of growth factor therapy with designed NGC scaffolds first requires an understanding of the underlying signalling arising from both, exposure to nerve growth factor (NGF) and topographical cues. Original findings revealed that increasing NGF levels in media combined with linear topography produced less dorsal root ganglia (DRG) axon network sizes, in contrast to larger such networks on flat substrates. This trend was confirmed and determined to start between day four and six in vitro. The interaction between growth factor and topographical signalling was found to be exclusive to the NGF signalling pathway with FGFb, BDNF not being affected. Application of blebbistatin, a myosin II inhibitor, caused network sizes of high NGF cultures to increase on microgrooved surfaces, becoming comparable to that of low NGF concentrations. Myosin IIA intensity analysis of growth cones showed, that higher NGF concentrations result in blocking of myosin IIA similar to blebbistatin. Morphologically, high NGF promoted densely fasciculated axon growth, low NGF showed a low, ordered fasciculation and blebbistatin application caused no fasciculation and highly branched morphology. Taken together; myosin IIA is concluded to be involved in suppressing fasciculation. The NGF trend was concluded to occur from extensive fasciculation induced adhesion loss, arising from the combination of blocking myosin IIA mediated defasciculation, microgroove confinement and greater neurite outgrowth. The implications of these findings are that support with and dosage of NGF may not be straight forward to support regeneration, if topographic guidance is provided at the same time.

Cell therapy is suggested in combination with NGC to provide a long-term supportive environment. To enable structure and guidance akin to bands of Büngner, here a sonotweezer device was used to fabricate aligned strings of adipose-derived-stem-cells (ADSC) embedded in a collagen scaffolds and its effectiveness for 3-D neurite guidance assessed. Bands of ADSC were formed inside collagen I hydrogels during gelation with different spacings (185 and 375µm) and using two cell concentrations at seeding (12.5 and 25k). ADSC generated contraction of the collagen matrices and this produced collagen fibre alignment. Aligned bands of ADSC contracted the gels perpendicular to the direction of the banding pattern; the degree of organisation of the collagen gels was dependent on the ADSC seeding density. DRG axons, regenerating through these cell seeded gels, were directed by the ADSC pattern.

To create mechanically anisotropic, cell-free materials that supported regeneration locally silk fibroin microfibres were embedded in collagen I hydrogels. Regenerating DRG axons were not guided if the silk fibroin microfibres were aligned. The silk microfibres were also used as a means to deliver NGF. The NGF loaded silk fibroin microfibres incorporated into collagen scaffolds was shown to promote greater neurite outgrowth compared to untreated silk fibroin microfibres, when NGF was not supplied with media. This approach may provide a non-cellular, biocompatible and biodegradable way to deliver growth factors locally.

This work has three major findings that provide the basis for future improvements to regenerative strategies for PNS repair: A) growth factor delivery may not always be beneficial and that particular combined therapies need to be investigated for potential interactions; B) cell therapy, as an alternative to growth factor delivery, may benefit from patterning approaches such as the sonotweezers devices used here, to support directed outgrowth; C) growth factor supplied locally by gel embedded reservoirs supports robust regeneration at a level similar to systemic administration.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Engineering and Physical Sciences Research Council (EP/F500424/1).
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Riehle, Dr. Mathis and Bernassau, Dr. Anne
• Date of Award: 2019
• Depositing User: Mrs Marie Cairney
• Unique ID: glathesis:2019-41141
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Apr 2019 10:30
• Last Modified: 03 May 2024 10:01
• Thesis DOI: 10.5525/gla.thesis.41141
• URI: https://theses.gla.ac.uk/id/eprint/41141