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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Weightman, Alan Peter (2015)
Languages: English
Types: Doctoral thesis
Subjects: QP
The extent of regeneration is often limited after spinal cord injury (SCI), due to the post-injury microenvironment that is unsupportive of nerve fibre regeneration and the limited intrinsic reparative capacity of neurons. Current mainstream clinical therapies focus on reducing the extent of damage in the early stages of injury, rather than promoting regenerative mechanisms in sites of pathology. In this context, one promising biomedical engineering strategy emerging globally to promote repair following SCI is the reconstruction of neural circuitry in injury sites via the implantation of polymer scaffolds, or ‘structural bridges.’ To date, the development of such synthetic bridges has faced two major challenges: an overwhelming reliance on basic 2-D scaffolds functionalised with single cell types (which therefore fail to mimic the complex circuitry of the neural lesion environment); and heavy dependence on live animal models of neurological injury for functional screening and developmental testing, in the absence of in vitro injury models that mimic the complex pathological sequelae of neurological injury in vivo.\ud \ud To this end, this thesis demonstrates an enhancement of the spatial and cellular complexity of both nanofibre-based scaffolds for spinal cord repair and in vitro SCI models for screening efficacious scaffold formulations. Nanofibre-hydrogel constructs containing aligned glial cell co-cultures (derived from primary sources) were successfully developed by systematically optimising the assembly protocol and construct design features. Further, protocols were developed to demonstrate the feasibility of increasing the number of constituent nanofibre layers in constructs with astrocyte mono-cultures, for further processing of constructs into an implantable form.\ud \ud A safe and effective method of inducing complete transecting lesions in organotypic spinal cord slice cultures was developed following the production of a prototype double-bladed lesioning tool. The development of quantitative image-based assays of fluorescently labelled astrocyte, microglial and neuronal cell populations within slice lesion sites showed mimicry of multiple cardinal features of neurological injury in vivo. Finally, a method was developed to coat portable frames of aligned nanofibres with therapeutic biomolecules and incorporate frames into lesioned slices. Patterns of nanotopography induced outgrowth/alignment of astrocytes and neurons in the in vitro model were strikingly similar to that induced by comparable materials in related studies in vivo. This demonstrates the predictive utility of the model and the potential to reduce and refine the use of lower-throughput live animal models for screening applications.
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