Abstract:
Spinal Cord Injury (SCI) is a traumatic disorder of the central nervous system (CNS) resulting in permanent sensorimotor functional deficits. Neurotrophins are a class of chemotrophic growth factors that have demonstrated potential to influence the trajectory of axonal outgrowth throughout development and following traumatic injury. Evidence has suggested that expression of these factors in the form of a concentration gradient may hold therapeutic benefit in SCI, by navigating spared axons towards pre-injury injury targets. In vitro models are crucial tools for investigating and optimising potential therapeutic compounds prior to testing within the in vivo setting. This thesis aimed to develop an in vitro model for investigating the effects of neurotrophic stimulation on neuronal cells. A protocol was developed and optimised to differentiate SH-SY5Y cells within a 96-well plate format to screen the effects of neurotrophins on neurite outgrowth. The effect of neurotrophins on neurite outgrowth was investigated in the presence and absence of chondroitin sulfate proteoglycan neurite outgrowth inhibitors, known to be involved in the pathophysiology of SCI. SH-SY5Y cells that were plated at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without affecting cell viability. Room temperature pre-incubation for 1 hour improved the plating homogeneity within the well and the ability to analyse neurites. Differentiated SH-SY5Y cells were successfully detached and re-plated, demonstrating their potential to be used as a model for axonal regeneration and guidance. Next, a compartmentalised gradient-generator was developed, and the concentration gradients quantified using FITC-dextran (10 kDa) as a model molecule to represent a neurotrophin. The concentration gradients of FITC-Dextran (10 kDa) were rapidly established within 1 hour. This gradient was sustained over a period of 96 hours with 24 hourly replenishment of source and sink reservoirs. Finally, agarose-gelatin bioinks were developed and characterised to evaluate their use for bioprinting concentration gradients as an alternative, more precise approach of gradient-generation. The developed bioinks possessed properties suitable for extrusion-based bioprinting and SH-SY5Y cell culture. Overall, the findings from this thesis contribute towards the development of a platform that can help researchers test new therapies for SCI and other neurological disorders.