Development and testing of chitosan micro-structured substrates for nerve regeneration

Peripheral nerve injuries are a common type of injury around the world, affecting more than 1 million people every year. Even though the intrinsic capacity of regeneration of the peripheral nervous system (PNS), the effective repair depends on the extent of the cut and the distance between the two ends of the nerve. In response to nerve injury, the Schwann cells undergo rapid changes in their phenotype, proliferate and migrate and provide a “pathway” (i.e. the Bungner bands) for the following axonal regrowth. This is a critical process for nerve regeneration. Nowadays there are still no efficient therapeutic treatments for this kind of condition and nerve grafts remain the gold standard in clinics. Recently, the use of hollow nerve guidance conduits has started. Chitosan, a chitin derived polymer, is a good biomaterial for tissue engineering applications and, in fact, basic plain chitosan nerve conduits have been approved for clinical use in Europe. In our body, cells can perceive physico-mechanical stimuli around them, and regulate their morphology, behaviour and fate in response to these stimuli. This process is generally referred to as mechanotransduction, where integrin receptors and focal adhesion pathway are the major molecular players. Several studies in vitro have demonstrated that neural cells cultured on nano/micro-structured substrates, such as directional gratings (GRs), respond to them, modifying their morphology and migration behaviour. The ability of anisotropic substrates to tune neural cells can be useful in the PNS regeneration framework, as devices patterned with directional micro-topographies could be used to orientate cells in the right direction and to achieve a more rapid and effective repair of the injured nerve. In this sense, patterning the inner part of hollow nerve guidance conduits can be an optimal way to enhance the effectiveness of these devices. In this thesis, chitosan based micro-structured substrates are produced and tested in vitro, with the aim to develop improved scaffolds for peripheral nerve regeneration. Here, we developed micro-structured substrates, made of chitosan, an FDA approved and biodegradable material, and enhanced with topography modifications, at micrometer level. We used three types of anisotropic patterns, presenting different levels of symmetricity: gratings (GRs), isosceles triangles (ISO) and scalene triangles (SCA), with a Flat surface as control. Line dimensions were 4 µm in width and 370 nm in depth. We developed an improved solvent casting method for the micro-structured chitosan films production. Moreover, we characterized their water absorption capacity and topography stability, showing overall good reproducibility levels for the procedure. The chitosan directional topographies were then tested in vitro with the RT4-D6P2T-GFP glial Schwann cells (a Schwannoma cell line). In general, the topographical modifications of the chitosan showed to improve Schwann cell adhesion and proliferation. We performed wound healing experiments (i.e. collective migration), in which cells showed an increased capacity to collectively migrate on the SCA pattern. Furthermore, cytoskeleton organization and cell-cell junctions were evaluated via immunostaining for actin fibres and N-cadherin. In general, we set up an improved method for the production of micro-structured chitosan films by solvent casting, with considerably reduced costs in term of time and materials used in the process and high fidelity in micro pattern reproducibility. In addition, films resulted more durable and their manipulation in the further biological experiments resulted easier. The cell experiments showed the impact of the directional patterns on the Schwann cells, showing that the directional asymmetric topographies (ISO and SCA) promote a faster migration of cells, both singularly and collectively. This effect was particularly enhanced on SCA patterns. In conclusion, the use of micro-structured directional topographies, such as SCA, may be exploited to enhance the nerve regeneration process mediated by chitosan scaffolds