Peripheral nerve injury as a result of trauma affects approximately 1 million people in Europe and America annually. The current clinical gold standard treatment for repairing long gaps is the nerve autograft, in which only -50% of cases result in satisfactory functional recovery. Tissue-engineered cellular bridging devices for surgical implantation into peripheral nerve injury sites could provide an attractive alternative to the autograft. This project reports the development of a robust, anisotropic biomaterial with highly aligned cells that can fonn the basis of a peripheral nerve repair device. Engineered neural tissue (EngNT), which is formed from columns of Schwann cells or stem cells within a 3D aligned collagen matrix, can promote directed neurite outgrowth in vitro. This study demonstrates that sheets of EngNT can be arranged to form the 'endoneurium' of a peripheral nerve repair device within a NeuraWrap™ outer tube, and can be used for the repair of critical sized defects in rat. Schwann cells are the preferred cell type for peripheral nerve repair because of their ability to enhance axon migration and secrete factors that further increase regeneration. However the use of autologous Schwann cells has a number of disadvantages, including the sacrifice of host nerve tissue for their extraction and slow expansion times in vitro. Various therapeutic cell types and a bovine collagen source that can potentially be used to make EngNT to form the device core were investigated. EngNT devices containing Schwann cell-like cells from adipose-derived stem cells (dADSC) or human neural progenitor cells differentiated to glial cells (dCX) were tested in a critical sized gap in the rat sciatic nerve model. The in vivo experiments demonstrated that there is potential, for the dADSCs to be used for peripheral nerve repair. The results from the dCX repairs were less clear. The technology reported here offers a simple, rapid and effective method for the manufacture of an aligned cellular biomaterial, and could be applied to a range of tissue engineering applications. This study demonstrates that there is potential for EngNT to be used in the construction of nerve repair conduits.

Nerve injury is a serious problem afflicting many thousands of patients annually. Despite advances in microsurgical treatments, functional recovery is often poor, and the development of new tissue engineered methods to repair nerves is an active area of research.

This project focused on the design of a cellular scaffold applicable for the promotion of peripheral nerve regeneration. Firstly, we established a correlation between the organization of HA/PEG co-polymeric networks to their mechanical and degradability properties; cell adhesion was conferred to all gels by the incorporation of RGD peptides. Three families of hydrogels were produced using different procedures to permit an increasing physical incorporation of HA into a PEGDA-based network. From a comparative study of rheological properties and enzymatic degradability, co-networks obtained using thiolated HA as chain transfer agent during PEGDA photo-polymerization were selected for further biological investigations, aiming to link the cellular response of L929 murine fibroblasts (phenotype, proliferation rate, metabolic activity) to the composition and the consistency of selected hydrogels. Our findings showed that there is a clear relation between increasing hardness and increasing cell spreading/proliferation rate. This study illustrated the possibility to fine tune cell/material interactions with appropriate reactive processing techniques. As a spin-off of this study, we become interested in the interplay of cellular interactions in the use of materials that contain both HA and RGD peptides, which can bind at the same time to HA receptors such as CD44 and av integrins. We focused on soluble HA derivatives, with or without dandling RGD peptides. The kinetics and the mechanistic details of both HA and HA-RGD internalization were studied in a phagocytic model (J774.2 murine macrophages). HA-RGD showed a form of synergic binding to integrins and CD44 (HA receptor), whereas its uptake remained solely regulated by CD44 dwell-time on the cell membrane. This study demonstrated that the knowledge of the rate-determining steps of the uptake of a carrier is necessary for assessing its efficiency. In this case, the presence of multiple ligands on a carrier was beneficial in some respect, but may not be optimal to overcome internalization limitations that arise from the slow turnover of the determining receptor. Finally, we studied the relation between the regulation of the expression of CD44 / RHAMM (HA receptor mediated motility) and the motility of Schwann cells (peripheral glial cells) and stem cells differentiated into a glial phenotype. Rt-PCR and immuno-assay experiments suggested that RHAMM up-regulation is associated with glial differentiation and we speculate that in the future this HA receptor could be considered as a differentiation marker. We also illustrated the importance of HA / RHAMM interaction for the motility of glial cells. These results indicate the importance of HA in mediating glial cell function during peripheral nerve regeneration and have implications for therapeutic repair strategies.