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.

Molecular sequelae of topographically guided peripheral nerve repair.

Peripheral nerve injuries cause severe disability with decreased nerve function often followed by neuropathic pain that impacts the quality of life. Even though use of autografts is the current gold standard, nerve conduits fabricated from electrospun nanofibers have shown promise to successfully bridge critical length nerve gaps. However, in depth analysis of the role of topographical cues in the context of spatio-temporal progression of the regenerative sequence has not been elucidated. Here, we explored the influence of topographical cues (aligned, random, and smooth films) on the regenerative sequence and potential to successfully support nerve regeneration in critical size gaps. A number of key findings emerged at the cellular, cytokine and molecular levels from the study. Higher quantities of IL-1α and TNF-α were detected in aligned fiber based scaffolds. Differential gene expression of BDNF, NGFR, ErbB2, and ErbB3 were observed suggesting a role for these genes in influencing Schwann cell migration, myelination, etc. that impact the regeneration in various topographies. Fibrin matrix stabilization and arrest of nerve-innervated muscle atrophy was also evident. Taken together, our data shed light on the cascade of events that favor regeneration in aligned topography and should stimulate research to further refine the strategy of nerve regeneration using topographical cues.

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.

Effect of modulating macrophage phenotype on peripheral nerve repair.

Peripheral nerve repair across long gaps remains clinically challenging despite progress made with autograft transplantation. While scaffolds that present trophic factors and extracellular matrix molecules have been designed, matching the performance of autograft-induced repair has been challenging. In this study, we explored the effect of cytokine mediated 'biasing' of macrophage phenotypes on Schwann cell (SC) migration and axonal regeneration in vitro and in vivo. Macrophage phenotype was successfully modulated by local delivery of either Interferon-gamma (IFN-γ) or Interleukin-4 (IL-4) within polymeric nerve guidance channels, polarizing them toward pro-inflammatory (M1) or pro-healing (M2a and M2c) phenotypes, respectively. The initial polarization of macrophages to M2a and M2c phenotype results in enhanced SC infiltration and substantially faster axonal growth in a critically-sized rat sciatic nerve gap model (15 mm). The ratio of pro-healing to pro-inflammatory population of macrophages (CD206+/CCR7+), defined as regenerative bias, demonstrates a linear relationship with the number of axons at the distal end of the nerve scaffolds. The present results clearly suggest that rather than the extent of macrophage presence, their specific phenotype at the site of injury regulates the regenerative outcomes.

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.

The use of lipid microtubes as a novel slow-release delivery system for laryngeal injection.

OBJECTIVES/HYPOTHESIS: The potential utility of direct injection of bioactive substances in the treatment of vocal fold tissue fibrosis is limited by rapid clearance from the injection site. The objective of this study is to evaluate the potential of a lipid-based microtube delivery system to preserve the biological activity of injected substances and prolong their duration of pharmacological effects in the larynx. STUDY DESIGN: Prospective in vitro and case-control in vivo murine study METHODS: Lipid-based microtubes were loaded with Texas red-dextran (MT-TR) and hepatocyte growth factor (MT-HGF). In vitro and in vivo (using a murine vocal fold injection model) release of MT-TR and MT-HGF were determined to assess duration of microtube-mediated delivery. The biologic effects of MT-HGF on fibroblasts were assessed after treatment in the presence of transforming growth factor (TGF)-ß. RESULTS: In vitro release kinetics demonstrated slow release of MT-TR and MT-HGF, correlating with in vivo results demonstrating persistence of MT-HGF at 4 weeks postinjection. Bioefficacy was maintained, as MT-HGF was shown to inhibit TGF-ß-mediated induction of procollagen mRNA levels in vitro 24 hours after treatment in fibroblast cells. Sustained release of HGF from microtubes at 6 days exacerbated the effects of TGF-ß and increased levels of procollagen mRNA. CONCLUSIONS: Microtubes have significant potential utility as an efficacious means of sustained-release delivery of bioactive agents to the larynx. Atthough the role of HGF as an antifibrotic agent is questioned, its sustained bioefficacy after microtube encapsulation distinguishes microtubes from other delivery vehicles.

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.

Advances in bioengineered conduits for peripheral nerve regeneration.

Although resorbable NGCs have been developed for peripheral nerve grafting, there has been little published on their use as a material for trigeminal nerve repair. Advances in engineered guidance channels and modifications to the single-lumen conduit with growth-permissive substrates, ECM proteins, neurotrophic factors, and supportive Schwann or stem cells, and anisotropic placement of these within the NGC may translate from animal models to clinical human use in the future. A great deal of research is still needed to optimize the presently available NGCs, and their use in peripheral trigeminal nerve repair and regeneration remains yet to be explored. Bioengineered NGCs and additives remain promising alternatives to autogenous nerve grafting in the future. They can incorporate all of the developing strategies for peripheral nerve regeneration that develop in concert with the ever-increasing understanding of regenerative mechanisms. The use of nanomaterials also may resolve the numerous problems associated with traditional conduit limitations by better mimicking the properties of natural tissues. Since cells directly interact with nanostructured ECM proteins, the biomimetic features of anisotropic-designed nanomaterials coupled with luminal additive ECMs, neurotrophic factors, and Schwann cells may provide for great progress in peripheral nerve regeneration.

Influence of nanoporous alumina membranes on long-term osteoblast response.

A major goal of bone tissue engineering is to design better scaffold configuration and materials to better control osteoblast behavior. Nanoporous architecture has been shown to significantly affect cellular response. In this work, nanoporous alumina membranes were fabricated by a two-step anodization method to investigate bone cell response. Osteoblasts were seeded on nanoporous alumina membranes to investigate both short-term adhesion and proliferation and long-term functionality and matrix production. Cell adhesion and proliferation were characterized using a standard MTT assay and cell counting. The total protein content was measured after cell lysis using the BCA assay. Matrix production was characterized in terms of surface concentrations of calcium and phosphorous, components of bone matrix, using X-ray photoelectron spectroscopy (XPS). The results from nanoporous alumina membranes were compared with those of amorphous alumina, aluminum, commercially available ANOPORE and glass. Results indicate improved osteoblast adhesion and proliferation and increased matrix production after 4 weeks of study.