Electrically conductive silk nanofibrous scaffolds with neuronal cell guidance cues for peripheral nerve regeneration

Abstract

Peripheral nerve injuries are a huge burden to society, and the therapies that are common treatments are still far from perfect. Hence, current research is exploring new, optimised therapies to treat these types of injuries. Nerve guidance conduits, which are synthetic devices that bridge the proximal and distal end of an injured nerve, are one of the most prominent ones. However, there are still challenges on current and future conduits, such as customisation, mimicking extracellular matrix properties, and microscale guiding of tissue regrowth. This thesis is focussed on the development of nanofibrillar silk fibroin devices that could impart microscale axonal guiding via customised topographies and morphologies; the fabrication of bioactive patterns; and determining the relationship between these two factors in neuronal development. Silk fibroin was electrospun using a combination of nanoyarn electrospinning and the use of sacrificial substrates to produce highly customisable seamless, hollow, aligned nanofibrillar tubes. By exploiting the modularity of the process, it was possible to manufacture multichanneled, seamless hollow conduits with a high degree of customisation in terms of number of microchannels, lumen diameter (192 to 652 µm), nanofibrillar alignment (10° to 40°), and wall thickness (15 to 35 µm). The mechanical properties of these devices are also very similar to the ones reported for human nerve tissue (elastic modulus of 22.2 ± 1.1 MPa, 50.6 ± 3.8 % strain to failure). To produce silk fibroin-based substrates that could become spatially-selectively bioactive, silk fibroin was functionalised with norbornene groups, which are candidates for UV-initiated thiol-ene reactions. The reactions were successful for both aqueous silk fibroin solutions, and for solid silk-fibroin electrospun mats. However, lyophilised functionalised silk solutions were not possible to be redissolved and produce electrospinning dopes. Solid-state functionalised silk fibroin mats presented a lower degree of functionalisation, but even with the lower degree of functionalisation, thiol-ene fluorescent patterns were achievable on the electrospun surfaces. Unexpectedly, the electrochemical conductivity of silk fibroin mats increased during the intermediate functionalisation and decreased after photopolymerising reflectin, a strong proton conductor. This behaviour was attributed to a change in the electrochemical charge, as norbornene-doped polymers are considered to be anionic conductors. These results imply that functionalised silk fibroin could be a good candidate to produce bioactive patterns through thiol-ene reactions. Finally, the cellular response of these silk fibroin-based materials was tested for both differentiated and undifferentiated NG108-15 neuroblastoma – glioma cells. It was observed that for undifferentiated cells, the driving mechanism to guide cell proliferation was mainly topographical, although biochemical cues also have a measurable influence. This influence, however, has not been found to be related to the electrochemical conductivity of the substrate, as both high and low conductive materials increase the directional proliferation. In terms of differentiated cells, no appreciable difference can be observed on the neurite length across the different substrates, while neurite alignment appears to be more closely linked to surface topography than to biochemical cues, with neurite angular spread distributions narrowing by at least 20° in every material. Overall, this study provides insights about the generation of optimised silk fibroin nanofibrillar conduits for nerve regeneration, and how biochemical modification of silk fibroin could be utilised to further enhance the topographical guidance provided by optimised nanofibrillar surfaces. Furthermore, the combination of both biochemical and highly optimised topographical cues could produce nerve guidance conduits with a higher axonal alignment and cellular proliferation in potential nerve regeneration therapies.

Date of Award	31 Jul 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sarah Cartmell (Co Supervisor), Eriko Takano (Co Supervisor) & Jonny Blaker (Main Supervisor)