Design and development of electrospun polymer substrates for neuronal cell culture and electrophysiological investigation

Repair of damaged or diseased regions of the central or peripheral nervous system will likely require stem cell treatment. Biomaterial substrates may be important in such neural tissue engineering applications to provide chemical cues to minimise glial scarring and provide both chemical cues and structural support for implanted and migrating cells as well as endogenous tissue. The nanostructure of such biomaterial substrates should mimic the extracellular matrix to ensure natural cell growth and functional development upon implantation. In mimicking the natural cellular environment, these biomaterial substrates are also strong candidates for three dimensional cell culture models, with such models likely to match the biological condition more closely and so produce more realistic and biologically relevant results than their two dimensional counterparts. Electrophysiological activity of neuronal cells is a critical determinant of neuronal communication to enable functional neuronal network formation. As such, it is critical that neurons cultured upon and within three dimensional biomaterial substrates be electrophysiologically tested to improve functional outcomes both in culture, and later upon implantation in regenerative neuroscience interventions. Previous studies have shown the potential of electrospun nanofibrous poly(-ε- caprolactone) substrates in both implantation and three dimensional neural (and other) cell culture applications. This project initially involved the development of degradable electrospun nanofibrous poly(-ε-caprolactone) substrates for three dimensional cell culture applications, in particular to enable electrophysiological characterisation of neuronal cells cultured upon them. Subsequently, assessment of the sensitivity and usefulness of such electrophysiological testing procedures for investigation of biomaterial substrates in three dimensional cell culture was assessed. Substantial optimisation of substrate design and preparation, development of novel inexpensive and repeatable cell-seeding techniques, optimisation of cell culture protocols, and design of appropriate controls to isolate effects of certain interrelated characteristics of electrospun substrates was required. These developments enabled long-term neuronal cultures for longitudinal functional studies, the first of any such study to be undertaken. Neuronal growth patterns in response to the nanoarchitectural topographical cues presented by electrospun polymer substrates were cell type dependant, with peripheral neurons extending neurites along substrate fibres, and central neurons from the hippocampus sending neurites both parallel and perpendicular to the fibres, mimicking expected natural in vivo growth patterns. Results also illustrated basic differences in neuronal cellular functional development with time in culture between peripheral sensory neurons from the dorsal root ganglion that were functional at all time points, and those of the hippocampus that did not reach functional maturity until 25 days in culture. Small increases in incubation temperature accelerated the degradation of poly(-ε-caprolactone) substrates, and inhibited functional development of neurons cultured on such substrates with time in culture, but not of cells cultured on glass. This presents the possibility that poly(-ε- caprolactone) may be problematic upon implantation where it is known that surgical sites can show elevated temperatures, albeit for restricted periods. This result illustrated the sensitivity of electrophysiological testing procedures, revealing diminished cellular functional development in response to accelerated poly(-ε- caprolactone) degradation resulting from nothing more than a poorly calibrated incubator, where growth studies of the same cells showed no effect of incubation temperature. From this, development of substrates for electrophysiological testing procedures demonstrated within this thesis present a sensitive method for assessing effects of biomaterials on functional outcomes in tissue engineering applications, particularly where degradable substrates are proposed.