Microchannel-based regenerative scaffold for chronic peripheral nerve interfacing in amputees.

Neurally controlled prosthetics that cosmetically and functionally mimic amputated limbs remain a clinical need because state of the art neural prosthetics only provide a fraction of a natural limb's functionality. Here, we report on the fabrication and capability of polydimethylsiloxane (PDMS) and epoxy-based SU-8 photoresist microchannel scaffolds to serve as viable constructs for peripheral nerve interfacing through in vitro and in vivo studies in a sciatic nerve amputee model where the nerve lacks distal reinnervation targets. These studies showed microchannels with 100 µm × 100 µm cross-sectional areas support and direct the regeneration/migration of axons, Schwann cells, and fibroblasts through the microchannels with space available for future maturation of the axons. Investigation of the nerve in the distal segment, past the scaffold, showed a high degree of organization, adoption of the microchannel architecture forming 'microchannel fascicles', reformation of endoneurial tubes and axon myelination, and a lack of aberrant and unorganized growth that might be characteristic of neuroma formation. Separate chronic terminal in vivo electrophysiology studies utilizing the microchannel scaffolds with permanently integrated microwire electrodes were conducted to evaluate interfacing capabilities. In all devices a variety of spontaneous, sensory evoked and electrically evoked single and multi-unit action potentials were recorded after five months of implantation. Together, these findings suggest that microchannel scaffolds are well suited for chronic implantation and peripheral nerve interfacing to promote organized nerve regeneration that lends itself well to stable interfaces. Thus this study establishes the basis for the advanced fabrication of large-electrode count, wireless microchannel devices that are an important step towards highly functional, bi-directional peripheral nerve interfaces.

Guiding intracortical brain tumour cells to an extracortical cytotoxic hydrogel using aligned polymeric nanofibres.

Glioblastoma multiforme is an aggressive, invasive brain tumour with a poor survival rate. Available treatments are ineffective and some tumours remain inoperable because of their size or location. The tumours are known to invade and migrate along white matter tracts and blood vessels. Here, we exploit this characteristic of glioblastoma multiforme by engineering aligned polycaprolactone (PCL)-based nanofibres for tumour cells to invade and, hence, guide cells away from the primary tumour site to an extracortical location. This extracortial sink is a cyclopamine drug-conjugated, collagen-based hydrogel. When aligned PCL-nanofibre films in a PCL/polyurethane carrier conduit were inserted in the vicinity of an intracortical human U87MG glioblastoma xenograft, a significant number of human glioblastoma cells migrated along the aligned nanofibre films and underwent apoptosis in the extracortical hydrogel. Tumour volume in the brain was significantly lower following insertion of aligned nanofibre implants compared with the application of smooth fibres or no implants.

Regenerative scaffold electrodes for peripheral nerve interfacing.

Advances in neural interfacing technology are required to enable natural, thought-driven control of a prosthetic limb. Here, we describe a regenerative electrode design in which a polymer-based thin-film electrode array is integrated within a thin-film sheet of aligned nanofibers, such that axons regenerating from a transected peripheral nerve are topographically guided across the electrode recording sites. Cultures of dorsal root ganglia were used to explore design parameters leading to cellular migration and neurite extension across the nanofiber/electrode array boundary. Regenerative scaffold electrodes (RSEs) were subsequently fabricated and implanted across rat tibial nerve gaps to evaluate device recording capabilities and influence on nerve regeneration. In 20 of these animals, regeneration was compared between a conventional nerve gap model and an amputation model. Characteristic shaping of regenerated nerve morphology around the embedded electrode array was observed in both groups, and regenerated axon profile counts were similar at the eight week end point. Implanted RSEs recorded evoked neural activity in all of these cases, and also in separate implantations lasting up to five months. These results demonstrate that nanofiber-based topographic cues within a regenerative electrode can influence nerve regeneration, to the potential benefit of a peripheral nerve interface suitable for limb amputees.

Photocrosslinkable chitosan based hydrogels for neural tissue engineering.

Hydrogel based scaffolds for neural tissue engineering can provide appropriate physico-chemical and mechanical properties to support neurite extension and facilitate transplantation of cells by acting as 'cell delivery vehicles'. Specifically, in situ gelling systems such as photocrosslinkable hydrogels can potentially conformally fill irregular neural tissue defects and serve as stem cell delivery systems. Here, we report the development of a novel chitosan based photocrosslinkable hydrogel system with tunable mechanical properties and degradation rates. A two-step synthesis of amino-ethyl methacrylate derivitized, degradable, photocrosslinkable chitosan hydrogels is described. When human mesenchymal stem cells were cultured in photocrosslinkable chitosan hydrogels, negligible cytotoxicity was observed. Photocrosslinkable chitosan hydrogels facilitated enhanced neurite differentiation from primary cortical neurons and enhanced neurite extension from dorsal root ganglia (DRG) as compared to agarose based hydrogels with similar storage moduli. Neural stem cells (NSCs) cultured within photocrosslinkable chitosan hydrogels facilitated differentiation into tubulin positive neurons and astrocytes. These data demonstrate the potential of photocrosslinked chitosan hydrogels, and contribute to an increasing repertoire of hydrogels designed for neural tissue engineering.

Role of fibronectin in topographical guidance of neurite extension on electrospun fibers.

Bridging of long peripheral nerve gaps remains a significant clinical challenge. Electrospun nanofibers have been used to direct and enhance neurite extension in vitro and in vivo. While it is well established that oriented fibers influence neurite outgrowth and Schwann cell migration, the mechanisms by which they influence these cells are still unclear. In this study, thin films consisting of aligned poly-acrylonitrile methylacrylate (PAN-MA) fibers or solvent casted smooth, PAN-MA films were fabricated to investigate the potential role of differential protein adsorption on topography-dependent neural cell responses. Aligned nanofiber films promoted enhanced adsorption of fibronectin compared to smooth films. Studies employing function-blocking antibodies against cell adhesion motifs suggest that fibronectin plays an important role in modulating Schwann cell migration and neurite outgrowth from dorsal root ganglion (DRG) cultures. Atomic Force Microscopy demonstrated that aligned PAN-MA fibers influenced fibronectin distribution, and promoted aligned fibronectin network formation compared to smooth PAN-MA films. In the presence of topographical cues, Schwann cell-generated fibronectin matrix was also organized in a topographically sensitive manner. Together these results suggest that fibronectin adsorption mediated the ability of topographical cues to influence Schwann cell migration and neurite outgrowth. These insights are significant to the development of rational approaches to scaffold designs to bridge long peripheral nerve gaps.