Isaac P. Clements

Thin-film enhanced nerve guidance channels for peripheral nerve repair

It has been demonstrated that nerve guidance channels containing stacked thin-films of aligned poly(acrylonitrile-co-methylacrylate) fibers support peripheral nerve regeneration across critical sized nerve gaps, without the aid of exogenous cells or proteins. Here, we explore the ability of tubular channels minimally supplemented with aligned nanofiber-based thin-films to promote endogenous nerve repair. We describe a technique for fabricating guidance channels in which individual thin-films are fixed into place within the lumen of a polysulfone tube. Because each thin-film is <10 microm thick, this technique allows fine control over the positioning of aligned scaffolding substrate. We evaluated nerve regeneration through a 1-film guidance channel--containing a single continuous thin-film of aligned fibers--in comparison to a 3-film channel that provided two additional thin-film tracks. Thirty rats were implanted with one of the two channel types, and regeneration across a 14 mm tibial nerve gap was evaluated after 6 weeks and 13 weeks, using a range of morphological and functional measures. Both the 1-film and the 3-film channels supported regeneration across the nerve gap resulting in functional muscular reinnervation. Each channel type characteristically influenced the morphology of the regeneration cable. Interestingly, the 1-film channels supported enhanced regeneration compared to the 3-film channels in terms of regenerated axon profile counts and measures of nerve conduction velocity. These results suggest that minimal levels of appropriately positioned topographical cues significantly enhance guidance channel function by modulating endogenous repair mechanisms, resulting in effective bridging of critically sized peripheral nerve gaps.

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.

Characterization of a composite injury model of severe lower limb bone and nerve trauma

Severe extremity trauma often results in large zones of injury comprising multiple types of tissue and presents many clinical challenges for reconstruction. Considerable investigation is ongoing in tissue engineering and regenerative medicine therapeutics to improve reconstruction outcomes; however, the vast majority of musculoskeletal trauma models employed for testing the therapeutics consist of single‐tissue defects, offering limited utility for investigating strategies for multi‐tissue repair. Here we present the first model of composite lower limb bone and nerve injury, characterized by comparison to well‐established, single‐tissue injury models, using biomaterials‐based technologies previously demonstrated to show promise in those models. Quantitative functional outcome measures were incorporated to facilitate assessment of new technologies to promote structural and functional limb salvage following severe extremity trauma. Nerve injury induced significant changes in the morphology and mechanical properties of intact bones. However, BMP‐mediated segmental bone regeneration was not significantly impaired by concomitant nerve injury, as evaluated via radiographs, microcomputed tomography (μCT) and biomechanical testing. Neither was nerve regeneration significantly impaired by bone injury when evaluated via histology and electrophysiology. Despite the similar tissue regeneration observed, the composite injury group experienced a marked functional deficit in the operated limb compared to either of the single‐tissue injury groups, as determined by quantitative, automated CatWalk gait analysis. As a whole, this study presents a challenging, clinically relevant model of severe extremity trauma to bone and nerve tissue, and emphasizes the need to incorporate quantitative functional outcome measures to benchmark tissue engineering therapies. Copyright

A conformable microelectrode array (cMEA) with integrated electronics for peripheral nerve interfacing

A high-resolution PDMS-based conformable microelectrode array (cMEA) with integrated electronics is implemented. The cMEA is incorporated into individual layers of a nanofiber-based nerve regeneration scaffold to create a novel regenerative electrode scaffold (RES) capable of establishing a stable, high-resolution peripheral nerve interface. The device features a compact size with an enhanced signal-to-noise ratio (SNR), as required by implantation applications. Preliminary characterizations of the device are performed using in vitro experimentations, including impedance spectroscopy and neural culturing.

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.

A regenerative electrode scaffold for peripheral nerve interfacing

Novel approaches to peripheral nerve interfacing are required to establish the stable, high-resolution connections demanded by the emerging generation of advanced neuroprosthetic devices. Here we propose a nanofiber scaffold-based design for a regenerative electrode capable of establishing significant numbers of stable and selective electrical connections with subsets of peripheral nerve. The design features one or more polyimide thin-film electrode arrays integrated within a layered nanofiber scaffold such that regenerating axons from a transected nerve are directed across the embedded electrodes. In-vitro and in-vivo experiments with a rat peripheral nerve model were performed to validate and optimize the ability of our regenerative electrode scaffold (RES) to direct axonal regeneration across an implanted electrode array. Immunostaining of cultured dorsal root ganglia revealed that migrating Schwann cells and extending neurites can be directed along oriented nanofibers and across an overlaid polyimide electrode in-vitro. RESs were then fabricated and implanted between the stumps of transected rat tibial nerves (n=10). After 3-6 weeks the scaffolds were explanted and stained to characterize regeneration through the RESs. Staining revealed robust axonal regeneration through the scaffolds. This regeneration was directed as close as several microns to the surfaces of the integrated electrode arrays. Staining also revealed minimal inflammatory response at the electrode array site. Additionally, the same results were obtained in the absence of an intact distal stump. In conclusion, our results suggest the feasibility of this design for use in interfacing an amputated nerve stump. Electrophysiological capabilities of the interface and facilitation of long term trophic support for the nerve will be examined in future experiments.

Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications

Microelectrode array (MEA) technology enables advanced drug screening and “disease-in-a-dish” modeling by measuring the electrical activity of cultured networks of neural or cardiac cells. Recent developments in human stem cell technologies, advancements in genetic models, and regulatory initiatives for drug screening have increased the demand for MEA-based assays. In response, Axion Biosystems previously developed a multiwell MEA platform, providing up to 96 MEA culture wells arrayed into a standard microplate format. Multiwell MEA-based assays would be further enhanced by optogenetic stimulation, which enables selective excitation and inhibition of targeted cell types. This capability for selective control over cell culture states would allow finer pacing and probing of cell networks for more reliable and complete characterization of complex network dynamics. Here we describe a system for independent optogenetic stimulation of each well of a 48-well MEA plate. The system enables finely graded control of light delivery during simultaneous recording of network activity in each well. Using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes and rodent primary neuronal cultures, we demonstrate high channel-count light-based excitation and suppression in several proof-of-concept experimental models. Our findings demonstrate advantages of combining multiwell optical stimulation and MEA recording for applications including cardiac safety screening, neural toxicity assessment, and advanced characterization of complex neuronal diseases.

Neuronal Tissue Engineering

The human nervous system gathers, transmits, processes, and stores information with remarkable speed and fidelity. These capabilities are made possible by networks of highly specialized neural cells, organized within an intricate three-dimensional framework of interconnected neural tissue. Only through a gradual and complex sequence of development does this neural tissue form and mature, in a process beginning soon after conception, and continuing long after the time of birth.

Microneedle cuff electrodes for extrafascicular peripheral nerve interfacing

OBJECTIVE The work presented here describes a new tool for peripheral nerve interfacing, called the microneedle cuff (μN-cuff) electrode. APPROACH μN arrays are designed and integrated into cuff electrodes for penetrating superficial tissues while remaining non-invasive to delicate axonal tracts. MAIN RESULTS In acute testing, the presence of 75 μm height μNs decreased the electrode-tissue interface impedance by 0.34 kΩ, resulting in a 0.9 mA reduction in functional stimulation thresholds and increased the signal-to-noise ratio by 9.1 dB compared to standard (needle-less) nerve cuff electrodes. Preliminary acute characterization suggests that μN-cuff electrodes provide the stability and ease of use of standard cuff electrodes while enhancing electrical interfacing characteristics. SIGNIFICANCE The ability to stimulate, block, and record peripheral nerve activity with greater specificity, resolution, and fidelity can enable more precise spatiotemporal control and measurement of neural circuits.Objective. The work presented here describes a new tool for peripheral nerve interfacing, called the microneedle cuff (μN-cuff) electrode. Approach. μN arrays are designed and integrated into cuff electrodes for penetrating superficial tissues while remaining non-invasive to delicate axonal tracts. Main results. In acute testing, the presence of 75 μm height μNs decreased the electrode-tissue interface impedance by 0.34 kΩ, resulting in a 0.9 mA reduction in functional stimulation thresholds and increased the signal-to-noise ratio by 9.1 dB compared to standard (needle-less) nerve cuff electrodes. Preliminary acute characterization suggests that μN-cuff electrodes provide the stability and ease of use of standard cuff electrodes while enhancing electrical interfacing characteristics. Significance. The ability to stimulate, block, and record peripheral nerve activity with greater specificity, resolution, and fidelity can enable more precise spatiotemporal control and measurement of neural circuits.