The development of a novel orientated nanofibre-containing hydrogel scaffold : in vitro studies of cell-substrate interactions and its application in vivo for peripheral nerve repair

Peripheral nerve injuries (PNI) causing gaps larger than a few millimeters require the interposition of a bridging material in order to allow nerve regeneration. The current gold standard treatment for such larger gaps, the autologous nerve graft, is associated with significant limitations and by no means guarantees a useful degree of functional recovery. Recent advances in tissue engineering have opened up new opportunities towards the development of alternative strategies to replace the autograft. The generation of complex three-dimensional (3D) scaffolds capable of mimicking the molecular and topographical cues of the extracellular matrix (ECM) found in native tissue is anticipated to improve the efficacy of simple hollow tubes that, so far, remain the only clinically-approved, tissue-engineered strategy for peripheral nerve repair. Despite intense research, tissue-engineered scaffolds are still incapable of out-perfoming the extent of repair supported by the autografted nerve and must be regarded as being far from satisfactory. For this purpose, the aim of the present study was to develop a novel biomimetic, nanofibre-based implantable nerve guide intended to enhance functional recovery after PNI. The first part reviews the theoretical background of biomaterials in the context of peripheral nerve repair. In particular, different aspects of the use of electrospun nanofibres such as the choice of the material, different approaches of surface functionalization and in vivo effects after implantation are discussed. The second part of the thesis addresses the development and in vitro characterisation of a 3D nanofibre-based fibrin hydrogel scaffold. The establishment of a protocol for the generation and hydrogel-embedding of multiple layers of orientated poly(ɛ-caprolactone) (PCL) nanofibres is demonstrated. A main focus is placed on cell-substrate interactions of highly purified populations of Schwann cells with the nanofibre and hydrogel components of their 3D environment. Embedded Schwann cells remain viable within the fibrin hydrogel for up to 7 days (the longest incubation time studied). Schwann cells within the fibrin hydrogel develop complex process branching patterns, but when presented with orientated nanofibres, show a strong tendency to redistribute themselves onto the nanofibres where they extend long processes that follow the longitudinal orientation of the nanofibres. The ability of this 3D, ECM-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggests that it may act as an appropriate device design for the bridging of experimental lesions of the peripheral nervous system (PNS). The third part of the thesis addresses the integration and performance of the novel nanofibre-based scaffold in an in vivo model of PNI. The novel conduits are assessed in a 15 mm rat sciatic nerve transection injury model and compared with simple entubulation, autograft and no repair using a wide range of morphological and functional methods. The presence of orientated nanofibres within the fibrin hydrogel promotes poor regeneration, however, when pre-seeded with isogenic Schwann cells, nanofibre-based conduits support nerve regeneration that is superior to simple entubulation repair in terms of the recovery of grip strength, the number of regenerated sensory dorsal root ganglia (DRG) neurons in retrograde tracing studies and the induction of compound muscle action potentials (CMAPs) recorded from the Musculus (M.) flexor digitorum brevis and the M. interosseus pedis dorsalis. Although the novel scaffolds were incapable of reaching the performance of autograft repair, future modifications offer several possibilities of optimizing scaffold characteristics to provide a better environment for nerve regeneration