Critical Reviews™ in Biomedical Engineering, 39(3):243–262 (2011)

Neural Tissue Engineering for Neuroregeneration and Biohybridized Interface Microsystems In vivo (Part 2) D. Kacy Cullen,1 John A. Wolf,1 Douglas H. Smith,1 Bryan J. Pfister2 Department of Neurosurgery, Center for Brain Injury & Repair, University of Pennsylvania, Philadelphia, PA; 2Department of Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ 1

Address all correspondence to���������������������������������������������������������������������������������������������������������������������� D.K. ��������������������������������������������������������������������������������������������������������������������� Cullen, Ph.D., University of Pennsylvania, Department of Neurosurgery������������������������������������������� , 105 ����������������������������������������� Hayden Hall / 3320 Smith Walk, Philadelphia, PA 19104; Tel: 215-746-8176; Fax: 215-573-3808; e-mail: [email protected].

ABSTRACT: Neural tissue engineering offers tremendous promise to combat the effects of disease, aging, or injury in the nervous system. Here we review neural tissue engineering with respect to the design of living tissue to directly replace damaged or diseased neural tissue, or to augment the capacity for nervous system regeneration and restore lost function. This article specifically addresses the development and implementation of tissue engineered three-dimensional (3-D) neural constructs and biohybridized neural-electrical microsystems. Living 3-D neural constructs may be “pre-engineered” in vitro with controlled neuroanatomical and functional characteristics for neuroregeneration, to recapitulate lost neuroanatomy, or to serve as a nervous tissue interface to a device. One application being investigated is developing constructs of axonal tracts that, upon transplantation, may facilitate nervous system repair by directly restoring lost connections or by serving as a targeted scaffold to promote host regeneration by exploiting axon-mediated axonal regeneration. In another application, living nervous tissue engineered constructs are being investigated to biohybridize neural-electrical interface microsystems for functional integration with the nervous system. With this design, in vivo neuritic ingrowth and synaptic integration may occur with the living component, potentially exploiting a more natural integration with the nonorganic interface. Overall, the use of tissue engineered 3-D neural constructs may significantly advance regeneration or device-based deficit mitigation in the nervous system that has not been achieved by non–tissue engineering approaches. KEY WORDS: neural engineering, neuroengineering, 3-D neural culture, axon, neuron, peripheral nerve injury, spinal cord injury, transplantation

I. Introduction The rapidly developing field of neural tissue engineering offers tremendous promise for future therapies to mitigate or reverse effects of disease, aging, or injury in the nervous system. Neural tissue engineering broadly spans a collection of many individual approaches (e.g., biomaterial scaffolds, biological grafts, stem cell therapy) for numerous nervous system applications (e.g., surgical, regenerative, disease treatment, deep brain stimulation). The central theme of this article on neural tissue engineering is the development and implementation of living, 3-D neural cellular based constructs.

In these applications, tissue engineered neural constructs consist of several combinations of living neurons distributed throughout neuronal specific matrices/scaffolds created in vitro prior to transplantation in vivo. Specifically, we review the development of living constructs engineered to (1) directly replace damaged or diseased neural tissue, (2) augment the capacity for nervous system regeneration and restore lost function, and (3) facilitate functional nervous system integration with living biohybridized neural-electrical microsystems. Neural tissue engineering design criteria are based on the unique application, ranging from

ABBREVIATIONS 3-D, three dimensional; PNS, peripheral nervous system; CNS, central nervous system; SC, Schwann cell; SEM, scanning electron microscopy; CAP, compound action potentials

0278-940X/11/$35.00 © 2011 by Begell House, Inc.

243

244 Cullen et al.

neuroregenerative to neural interface modalities in vivo. The complexity of these 3-D applications makes it difficult to design and assemble solutions within the living body. In contrast, engineering these 3-D systems in vitro results in a level of control not possible in vivo. Characteristics such as the cellular composition, matrix/scaffold properties, mechanical environment, and exogenous factors may be by design. For instance, matrix gelation behavior, charge gradients, and/or pressure gradients may be exploited to engineer constructs with cells dispersed throughout a 3-D matrix, either in a homogenously (random) or a choreographed fashion (discrete neuronal populations and/or segregating cell types). These systems are also amenable to control of the physical environment of the cells through the application of external forces, which we exploit to drive and control neural cell growth. This review addresses key design considerations and criteria based on particular studies or applications, specifically engineered constructs for neuroregeneration and biohybridized neural interface microsystems. For neuroregenerative applications, neural constructs may be “pre-engineered” in vitro with controlled neuroanatomical and functional characteristics based on the desired application. In particular, a common feature in many neurological diseases or trauma is the loss of axons, typically long projections connecting populations of neurons or relaying peripheral signals. Axonal regeneration is often insufficient due to long distances to appropriate peripheral targets1–5 or the nonpermissive environment of the central nervous system.6–8 Overall, living tissue engineered axonal constructs may be a promising strategy to facilitate nervous system repair by directly restoring lost connections or by serving as a scaffold to promote host regeneration by exploiting axonmediated axonal regeneration. Three-dimensional neural tissue engineering technology can also be applied to functionally integrate neural cells with engineered nonbiological components for electrical or electrochemical communication. Such biohybridized neural interface microsystems may be designed for sustained neural-electrical interface with the peripheral nervous system (PNS) or

central nervous system (CNS).9,10 The concept of “pre-engineering” 3-D tissue engineered constructs in vitro for a particular neurological disease/injury treatment is an extremely valuable approach. While it offers the advantage of experimental control, a clear understanding of design parameters and their influence on cell survival, growth, and functionality is necessary for successful application. Important considerations for future clinical applications of these technologies include adequate survival (e.g., immune tolerance, mass transport) and functionality (e.g., avoidance of aberrant connectivity) to achieve desired functional restoration. Collectively, these observations will present the major challenges as well as the key factors for successfully engineering these constructs. II. Neuroregeneration In vivo II.A. Overview Neural tissue engineering is continuously applying new solutions for nervous system repair and regeneration. Most strategies individually focus on novel biomaterials, trophic support, or cellular therapies. Pure biomaterial strategies (e.g., acellular scaffolds) are appropriate in many scenarios and are intended to recruit endogenous cells to form new tissue in vivo.11,12 While trophic support is important, successful regeneration will be achieved in combination with other strategies.2,6,13,14 Engineered cells and/or cell-replacement strategies (stem cells in particular) are typically delivered directly to the nervous system (see Ref. 15 for a review). While each individual strategy has shown promise, investigators in the field of neural tissue engineering recognize that it will be a combination of strategies that provides the best solution to nervous tissue repair.5,6,8,13,16–19 Evolving neural tissue engineering technology involves the integration of engineered living tissue constructs to directly replace lost function and/or to facilitate and augment the capacity for host nervous system regeneration. The key attributes of tissue engineered constructs are neural cells (oriented or random), three-dimensionality, and a scaffold support structure. Typically, tissue engineered constructs are completely or partially created in vitro, Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

with final 3-D form sometimes occurring in vivo. Depending on the desired function, the integration with this engineered living tissue may be temporary or permanent, including attenuating degeneration, augmenting endogenous regeneration, or serving as a permanent cell/tissue replacement. Overall, these 3-D neural constructs may be engineered in vitro prior to transplantation in vivo with controlled physical and functional characteristics to combat specific disease or injury conditions. II.B. Applications Many forms of nervous system trauma or disease share a common feature of loss of axons, often involving the long projections connecting populations of neurons in the brain and spinal cord or relaying peripheral sensory/motor signals. Accordingly, a common goal in neurorepair and neural tissue engineering is to promote and direct axonal regeneration. Examples of such conditions include physical trauma (e.g., stroke, spinal cord injury), tumor resection (e.g., acoustic neuroma, optic nerve meningioma), and neurodegenerative diseases (e.g., Parkinson’s disease, multiple sclerosis).20–24 Depending on the circumstances, this axonal loss may occur with or without associated perikaryal death.25–28 However, in the CNS, appropriate axonal regeneration does not occur due to a combination of a nonpermissive microenvironment, extraordinary distances to appropriate targets, and the potential for aberrant synaptogenesis (see Refs. 6, 13, 29–31 for reviews). To combat these problems, strategies to coax long-distance, targeted axonal connections are actively being pursued. For example, following injury, strategies involve both enhancing the intrinsic ability of axons to regenerate32–34 and modifying extrinsic factors to create a microenvironment more permissive for axonal outgrowth.35–37 Biomaterial-based strategies with micro- and nanoscale features are being investigated to promote axonal regeneration. In particular, tubular guidance conduits are being developed to facilitate repair following spinal cord injury38–41 or peripheral nerve injury.5,42–44 Engineered nano- and/or microfibers have shown promise by directly or indirectly eliciting robust and longitudinal axonal outgrowth Volume 39, Number 3, 2011

245

in vitro42 and in vivo.45,46 The addition of anisotropic topology and growth-promoting cues has proven to enhance neurite outgrowth in vitro47 as well as repair and regeneration in vivo.48 Moreover, conduits consisting of aligned microchannels have been engineered for the specific purpose of promoting Schwann cell (SC) infiltration and organization, thus facilitating longitudinal axonal regeneration in the PNS.49 Similarly, nerve guidance channels are also being developed that contain SCs to promote axon growth; however, these cells are typically not organized prior to implantation.50 For reestablishment of proper connectivity, precise spatial presentation of growth promoting as well as inhibitory signals will be required, as was demonstrated in targeting sensory axon regeneration to synapse at appropriate targets in the spinal cord.51 In addition, cell transplantation strategies are promising due to the ability to provide trophic support while potentially replacing a range of cell types lost due to trauma or disease.52–56 However, seldom do these potential therapies deliver neural cells preformed into a particular architecture capable of recapitulating longer segments of lost neuroanatomy. Despite several promising strategies actively being pursued, the goal of long-distance, targeted reinnervation between discrete nuclei remains elusive.2,5,12 Alternatively, the transplantation of pre-engineered living axonal tracts projecting from differentiated neurons may be an attractive solution to directly reconnect discrete neuronal populations in the central nervous system. Based on prescribed functional characteristics (e.g., neuronal subtype, genetic modification), these living axonal tracts could potentially “wire in” to directly restore lost function, thereby reconnecting previous synaptic partners or creating new circuits around a damaged region. Alternatively, such axonal constructs may promote and guide axonal regeneration by exploiting axon-mediated axonal outgrowth. As such, axonal constructs may provide a living scaffold for directed axonal regeneration, leading to targeted nerve tract reestablishment and synaptic integration with final end targets (e.g., other neuron(s), muscle, sensory organ). One mechanism by which axonal constructs

246 Cullen et al.

FIGURE 1. Three-dimensional constructs for neuroregeneration and biohybridization. (a–b) Tissue engineered constructs were “pre-engineered” in vitro to contain long axonal tracts, thus recapitulating the architecture of lost neural tissue to directly replace lost axonal tracts or to serve as living targeted scaffolds for axonal regeneration. Constructs may be (a) unidirectional or (b) bidirectional, depending on the application specific to trauma or disease condition. (c-d) Biohybridized neural interface microsystems formed around 3-D electrodes create unique microsystems that are powerful platforms for enhancing the interface with the nervous system. (c) Cells can be grown in vitro on electroconductive microfibers and encapsulated with hydrogel or (d) the microfibers can be incorporated with the axonal constructs prior to transplantation.

promote targeted, expeditious regeneration may be axon-mediated axonal outgrowth. Axon-mediated axonal outgrowth has been studied in the context of developmental neurobiology, but the mechanism and potential for this mode of axon outgrowth for regeneration is unknown. Alternatively, the potential for glial-based axonal regenerative guidance has been established. For instance, in peripheral nerve regeneration, the glia, referred to as Schwann cells, are necessary to facilitate axonal regeneration

through alignment and the formation of the Bands of Bungner.57 Recently, the ability of astrocytes to mediate aligned neuritic outgrowth was demonstrated, which required an anisotropic alignment of astrocytic cell bodies and processes.58 Axon-mediated axonal regeneration may prove to be equally robust. This combination of anisotropic contact guidance and neurotrophic support would provide a labeled pathway for axonal outgrowth, potentially facilitating expeditious, targeted regeneration. Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

However, further understanding of mechanisms driving axon-mediated axonal outgrowth may be useful when applied to tissue engineered constructs designed to promote directed axonal regeneration following injury. II.C. Tissue Engineered Living Axonal Constructs Here we describe two fundamentally different strategies to create living 3-D neural tissue constructs “pre-engineered” in vitro to recapitulate the neuroanatomy lost due to disease or trauma (Fig. 1). These constructs are designed to serve as neural tissue replacements to recapitulate lost axonal tracts that, upon transplantation, may restore function by direct replacement and/or serving as a scaffold for targeted reconnection. The first strategy consists of long tracts (e.g., several centimeters long) of mechanically engineered living axons encapsulated in 3-D matrices. In this approach, long axonal tracts are generated by a newfound process of “axon stretch growth,” the mechanisms and applications of which have recently been described.59–61 In a second strategy, tubular microconduits (e.g., 500-µm diameter) with internalized living longitudinally aligned axons were engineered to be delivered into the nervous system in minimally invasive fashion for targeted restoration of axonal tracts. Axonal constructs engineered via stretch-growth in vitro. The central feature of these tissue engineered constructs is tracts of living axons created in vitro by the controlled separation of two integrated populations of neurons60,62 We have designed and built a series of axon elongation devices for the purpose of creating nerve constructs in vitro.61,63 We designed the devices to physically split integrated neuronal cultures into two halves and progressively separate the halves further apart using a microstepper motor system that can operate within a cell culture incubator. Accordingly, bundles of axons that crossed the border between the two halves prior to separation would be progressively elongated (Fig. 2). We have demonstrated and optimized extreme stretchgrowth of integrated axonal tracts using controlled mechanical tension, and have used various neuronal subtypes including cerebral cortical neurons, Volume 39, Number 3, 2011

247

neuronal-like cell lines, and dorsal root ganglion neurons. Using this technique we have grown tracts of up to 10 cm in length containing over a million axons.60 Scanning electron microscopy (SEM) studies of stretch-grown axons showed that the external properties of the rapidly elongating axon bundles appeared healthy and robust, with fascicles of tightly joined axons following an amazingly straight path (Figs. 2d, 2e). Indeed, stretch-growth of axons is well tolerated, producing a normal appearing cytoskeleton, and retaining the ability to transmit electrophysiological signals.64 Exploiting this axonal stretch-growth technology, we have recently developed a novel tissue engineering approach to create transplantable living nervous tissue constructs composed of parallel tracts/fascicles of axons spanning two neuronal populations. Prior to transplantation, the stretchgrown axonal tracts were embedded in a proteinaceous matrix and removed from the culture environment en masse.61,63 For the application of PNS repair, these engineered nervous tissue constructs consisting of living axonal tracts mimic the uniaxial geometry of axons in the missing nerve segment, and thus may facilitate axonal regeneration (Fig. 3). Recently, we utilized the resulting 3-D tissue engineered stretch-grown axonal constructs to bridge an excised segment of peripheral nerve in the rat.65 The axonal constructs were transplanted to bridge an excised segment of sciatic nerve (>1 cm) in the rat and histological analyses were performed at time-points up to 16 weeks post-transplantation to determine graft survival, integration, and host regeneration. We observed tissue engineered axonal constructs with surviving clusters of graft neuronal somata at the proximal and distal extremes of the constructs spanned by long axonal tracts; thus the overall geometry was maintained (Fig. 3). In addition, we have found comprehensive integration of the transplanted ganglia and axons with the host axons. In particular, throughout the transplanted region, there was an intertwining plexus of host and graft axons, suggesting that the transplanted axons mediated host axonal regeneration across the lesion (Fig. 4). By 16 weeks post-transplant, extensive myelination of axons was observed throughout

248 Cullen et al.

FIGURE 2. Axonal constructs engineered via stretch-growth in vitro. (a) Stretch-growth is induced by the controlled separation of two integrated populations of neurons over a period of days (adapted with permission from Ref. 65, Mary Ann Liebert, Inc.). (b-e) Bundles of axons that crossed the border between the two halves prior to separation are progressively elongated, leading to fasciculated tracts of hundreds of axons. (b-c) Confocal immunocytochemistry for tubulin (bar = 50µM) and (d-e) electron micrographs. (b-c) Reproduced with permission from Ref. 62, Mary Ann Leibert, Inc. (d) Adapted with permission from Ref. 61, Elsevier. (e) Adapted with permission from Ref. 60, Society for Neuroscience, Highwire Press. Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

the transplant region. Furthermore, graft neurons had extended axons beyond the margins of the transplanted region, penetrating into the host nerve. Notably, these allogenic transplanted neurons/axons survived at least 16 weeks post-implant in the absence of immunosuppression. The apparent immuno-privilege of our transplanted stretch-grown axonal constructs in the PNS challenges the conventional wisdom of transplantation rejection, leading us to postulate that the pure neuronal phenotype, the long axonal segments, and/ or rapid integration with host axons facilitated survival. Finally, at 16 weeks post-implant, compound action potentials (CAP) were measured across this transplanted nervous tissue bridge in all animals that were repaired using the living nerve construct (no action potentials were elicited in animals that had been transected without repair). These findings demonstrate the promise of living tissue engineered axonal constructs to bridge major nerve lesions and promote host regeneration, potentially by providing axon-mediated axonal outgrowth and guidance. As a next step, these constructs can be grown to be several centimeters long, appropriate for major nerve injuries. Notably, similar tissue engineered constructs consisting of living axonal tracts were successfully applied to repair spinal cord injury.63 Living axonal microconduits. In a forward-looking application for CNS repair, we are developing microengineered 3-D hydrogel microconduits containing a discrete neuronal population with internalized living axonal tracts extending unidirectionally several millimeters through the interior.66 These micro–tissue engineered conduits are approximately 500 µm in diameter—roughly three times the average diameter of a human hair—and up to several centimeters in length. The small size enables delivery into the brain or spinal cord via stereotaxic microinjection. Thus, these living axonal microconduits were specialized for the application of reconnecting discrete neuronal populations in the brain or spinal cord to restore function following disease or trauma. In principle, these 3-D engineered microconduits may potentially serve the dual purpose of a neuronal/axonal replacement with pre-engineered cytoarchitecture to directly reVolume 39, Number 3, 2011

249

store lost connectivity, as well as providing a living labeled pathway to facilitate targeted host regeneration if the source neurons are intact and capable, especially in cases where long-distance axonal regeneration is not feasible. Moreover, living 3-D axonal constructs may provide a regenerative path to bridge nonpermissive and/or inhibitory barriers such as the glial scar which commonly forms following trauma. The overall strategy is to create neural tissue engineering constructs “pre-engineered” in vitro to contain living aligned axonal tracts, thus mimicking a key feature lost to disease or trauma. Moreover, living axonal conduits may be unidirectional or bidirectional (Fig. 1), and may employ alternate geometries to enable some degree of curvature. These axonal conduits may then be contained within nerve guidance channels to protect the axonal tracts during and after implantation. The key to applying this strategy is the ability to generate long (up to several centimeters) living axonal tracts, and the capability to create a 3-D architecture to provide support prior to delivery in vivo. Moreover, the microversions of this technology were developed specifically to be delivered in a minimally invasive fashion without damaging the transplanted neurons and the axonal architecture. Overall, these living axonal constructs exploit tailored physical properties, anisotropy, and contact guidance to promote expeditious and targeted reinnervation. These engineered transplantable living axonal constructs may significantly add to the repertoire of tissue engineered strategies for nervous system repair. By recapitulating the neuroanatomy of tissue lost due to trauma or disease, these axonal tracts may serve as functional replacements or a labeled pathway to guide host axonal regeneration, potentially exploiting axon-mediated axonal regeneration. However, there are key challenges related to survival and functionality that require further study. Acute transplant survival will be influenced by mass transport, after which short- and long-term immunological responses will be critical. It is noteworthy that if the goal of transplanted axonal constructs is to provide a living labeled pathway for host axonal regeneration, then the graft may need to survive for

250 Cullen et al.

FIGURE 3. Neuronal survival and maintenance of architecture in engineered nervous tissue constructs. Representative confocal reconstructions of transplanted GFP+ engineered nervous tissue constructs used to bridge an excised segment of sciatic nerve (6 weeks post-implantation). (A) Continuous proximal (top) and distal portions (bottom) from a GFP+ construct immunolabeled for NF-200 (red) (scale bars = 0.5 mm). Multiple transplanted ganglia were evident on the proximal and distal ends (arrowheads) with aligned axonal tracts spanning these neuronal populations. Remnants of the PGA tube were observed bordering the transplant at this timepoint (note arced border material autofluorescing red). (B-C) Higher magnification regions from (A) rotated 90° (scale bars = 100 µm). (B) Major bundles of neurites projected from the proximal ganglia across the constructs as well as into host nerve towards the spinal cord (white arrow). (C) Similarly, neuritic bundles also projected from the distal ganglia across the constructs and distally into the distal nerve segment (yellow arrow). (D-F) Increased magnification from specified regions; GFP+ (left column), NF-200+ (center column), with overlay (right column); scale bars = 20 µm. (D) Central axonal tracts co-labeled for GFP and NF-200. (E) Transplanted ganglia became dense, three-dimensional clusters of neurons. (F) Neurites growing from the host into the proximal end of the constructs were observed as NF-200+ axons that were not co-localized with GFP (arrows). Reproduced with permission from Ref. 65, Mary Ann Leibert, Inc.

Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

251

only the time it takes the host axons to grow across. In addition, in cases where transplanted neurons/ axons assume a permanent functional role within the host nervous system, issues related to proper connectivity, plasticity, and avoidance of deleterious connectivity will be paramount. III. Biohybrid Neural Interface Microsystems In vivo III.A. Overview Each year in the U.S., several hundred thousand people suffer debilitating injuries that ultimately result in the loss of limb function. These populations would be greatly served by a new generation of prosthetic devices. The ideal neuroprosthesis would be a functional facsimile of the amputated limb, and facilitate continuous bidirectional communication between the CNS and the external environment. Currently, the vast majority of efforts in this area focus exclusively on the reestablishment of motor control, relying on visual feedback to guide the movement of the prosthesis. However, in order to achieve truly “normal” interaction with the surroundings, tactile feedback is vital. Additionally, from a clinical and rehabilitation standpoint, it is important to have an architecture that minimizes surgical complexity and recovery time, provides a hospitable environment for nerve survival, and lends itself to rapid learning. In the drive towards a fully functional neuroprosthesis, efforts are being made in the fields of both invasive and noninvasive neural interfaces, with only moderate success thus far. Although these techniques are able to restore some function, they remain cumbersome, often involve complex surgical procedures, and are limited in the versatility and complexity of functions that they can perform.67–73 Moreover, for these approaches it is challenging to enable afferent signaling of sensory stimuli directly to the nervous system. It is evident that an urgent need exists for a versatile neural interface that can enable bidirectional communication with prosthetic devices, while minimizing surgical complexity and time for recovery. However, there is currently no approach that directly innervates the nervous sysVolume 39, Number 3, 2011

FIGURE 4. Host axons growing along transplanted axons. Axons from the surviving cluster of transplanted neurons at the graft interior (blownup region from Fig. 3). Axons from the transplanted neuronal constructs are labeled green (GFP+) and transplant and host neurofilament-positive axons are immunostained red. These axons are a mix of the transplanted axons and host axons, suggesting that host axonal growth occurs via axon-mediated axon regeneration. Adapted with permission from Ref. 65, Mary Ann Leibert, Inc.

tem while preserving the totality of the processing power of the brain and spinal cord. Current neural-electrical interface platforms have been developed for extracellular monitoring and modulation of neuronal population activity over weeks, months, or even years. For interfacing with the nervous system of living organisms, arrays of sharp penetrating electrodes have traditionally been used to record neuronal population signaling. These systems have classically involved rigid metal and/or

252 Cullen et al.

FIGURE 5. Host axonal growth near transplanted multielectrode array (MEA). Host axonal ingrowth towards the MEA was observed via immunohistochemistry for tau (red). Note numerous tau+ axons within tens of microns of the MEA (partially sectioned during tissue processing). Adapted with permission from Ref. 10, Maney Publishing.

Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

silicon electrodes. However, there is typically signal attenuation or degradation over time, which may be attributed to a general foreign-body and inflammatory response resulting in localized scar tissue and a decrease in the neuronal density in the vicinity of an electrode.74,75 Thus, strategies to mitigate the detrimental response to chronic electrode placement are actively being pursued. For instance, there has been recent interest in developing more flexible electrodes (Fig. 5) to mitigate detrimental effects due to the mismatch of mechanical properties.76 Future approaches might combine neural tissue engineering with electrical interface technology to develop a strategy to coax axonal or dendritic growth from the host to the interface hardware for functional integration. The resulting biohybridized neural interface microsystems would serve as tissue engineered neural-electrical relays for improved neural integration and chronic performance. Also, in specific cases, this technique may provide a replacement end target for nervous system integration in cases where the original target is missing or irreparably damaged. These techniques may be useful for chronic integration with the CNS (e.g., retinal prosthesis). However, we focus on applying these biohybridized microsystems for integration with the PNS (e.g., to ultimately drive a robotic prosthetic limb), which we view as the “back door” into the brain. III.B. Biohybrid Interface Microsystems for Functional Integration with Peripheral Nerve Biohybrid neural interface microsystems have tremendous potential as constructs to augment in vivo neuroregenerative capabilities while enabling a living biohybridized interface with the nervous system. The key concept underpinning this biohybridized microsystem is to enable robust host axonal integration, thus providing the infrastructure to acquire motor output and enabling sensory input. In this particular case, it is surgically and computationally advantageous for the neural interface to occur with the PNS to avoid implanting electrodes into the otherwise noninjured brain or spinal cord, while simultaneously being at the point of final brain output [receiving motor command signal(s) Volume 39, Number 3, 2011

253

for actuation] and primary sensory input (sensory and proprioceptive feedback). However, peripheral nerve axons require a living target for innervation, hence the use of our biohybridized living tissue engineered relays. Because host integration may occur with the biological component of this biohybrid platform, this may exploit a more natural interface. In theory, this approach leverages the exquisite processing capacity of the CNS rather than straining to decipher it. Also, this strategy may circumvent the issue of scar tissue formation in the brain that is thought to be primarily responsible for the relatively short windows of operation for conventional electrodes. Thus, this approach may be promising to advance prosthetic control by exploiting a more natural interface, potentially enabling substantial complexity of the command signal while providing a vehicle for proprioceptive and other sensory feedback. Our current efforts are to develop and implement nervous tissue interface platforms consisting of arrays of mechanically compliant electrodes embedded in living 3-D neural cellular constructs. Thus, our goal is to engineer 3-D neural cellular constructs,10,61,77 with the added element of electrical functionalization via construct formation around a conductive backbone. An additional advantage of these neurobiologically active living tissue engineered neural relays is that a stable electrical interface may be formed in vitro prior to implementation in vivo. Host integration may occur with the biological component of this biohybrid platform, thus potentially leading host axons to electrically active components.10,77 Forming stable interfaces in vitro prior to in vivo integration may also mitigate several of the factors believed to contribute to performance degradation of chronically implanted electrodes.78 1. Neural-Electrical Relays using Electrically Active Fiber Arrays In one strategy, we are engineering living tissue engineered neural-electrical relays through the development of custom-built, encapsulated nervous tissue interface platforms consisting of arrays of mechanically compliant microelectrodes embedded in living

254 Cullen et al.

3-D neural cellular constructs. Such technology may serve as functional components for a biohybrid neural interface microsystem to be used as quantitative neurophysiological platforms or as neurobiologically active electrical relays. Electrically conducting polymer fibers are attractive substrates for sustained functional interfaces with neuronal populations due to their relative flexibility (compared to metal or silicon), modifiable geometry and chemistry, and controlled electroconductive properties.79,80 Polyaniline-based electrically conducting polymer fibers are attractive substrates for sustained functional interfaces with neurons due to their flexibility, tailored geometry, and controlled electroconductive properties. To date, we have addressed the neurobiological considerations of utilizing small diameter (<400 µm) fibers consisting of a blend of electrically conductive polymers as the backbone of encapsulated tissue engineered neural-electrical relays.9 We devised new approaches to promote survival, adhesion, and neurite outgrowth of primary dorsal root ganglion neurons on the fibers. We attained a greater than tenfold increase in the density of viable neurons on fiber surfaces, to approximately 700 neurons/mm2, by manipulating surrounding surface charges to bias settling neuronal suspensions towards fibers coated with cell-adhesive ligands (Fig. 6). This stark increase in neuronal density resulted in robust neuritic extension and network formation directly along the fibers. Additionally, we encapsulated these neuronal networks on fibers using agarose to form a protective barrier while potentially facilitating network stability. Following encapsulation, the neuronal networks maintained integrity, high viability (>85%), and intimate adhesion to PA-PP fibers (Fig. 7). These efforts accomplished key prerequisites for the establishment of functional electrical interfaces with neuronal populations using small-diameter electrically active fibers, including high-density neuronal adhesion and neuritic network development directly on fiber surfaces. 2. Neural-Electrical Relays with StretchGrown Axons We have designed and optimized techniques to adapt our stretch-grown axonal constructs for sta-

ble use with flexible multielectrode array technology.10 Both stretch-grown and non-stretch-grown cultures were maintained over several days in vitro with one end pre-adhered to a multielectrode array. Then, the neuronal/axonal cultures were encapsulated and removed from the culture environment en masse. Following insertion into a surgical tube, the constructs were attached to the proximal stump of a transected peripheral nerve in the rat. Over weeks post-implantation, the proximal nerve remained in intimate contact with our electrode array within our construct. Moreover, we observed evidence of host revascularization into the constructs. We utilized standard tissue processing techniques and immunohistochemistry to assess host regeneration using antibodies for the axonal protein tau. Remarkably, we found host axons had regenerated within the constructs and were within tens of microns of the implanted electrode array (Fig. 5). These results provide strong evidence for the feasibility of this approach to form a neural-electrical interface with spared segments of the PNS following trauma. Thus, we have made substantial advancements in implementing these living constructs in 3-D tissue engineered neural-electrical relays incorporating compliant electrode array technology.10 These techniques are complimentary to our methodology to grow high-density neuronal networks directly on conductive microfibers with subsequent hydrogel encapsulation in preparation for in vivo transplantation.9 This technology may have dual integrative ability: providing a surrogate end target in cases where the original target is irreparably damaged or lost and/or promoting axon growth to integrate with the electrodes by providing a living scaffold. Moving forward, a key challenge will be the demonstration of synaptic integration of the regenerating sensory and motor peripheral axons with our biohybridized microsystems, which may be necessary for chronic integration and function. IV. Summary and Challenges Research in neural tissue engineering and biohybrid neural interface microsystems is driven by the development of cutting-edge regenerative medicine and interface technology. A promising approach is Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

255

FIGURE 6. Controlled neuronal adhesion to conductive polymer microfibers. Confocal reconstructions of neuronal cultures plated on microfibers immunolabeled at 7 days in vitro for MAP-2 (green) and tau (fiber locations denoted by dashed lines). By controlling the relative electrostatic surface charge of the microfiber and the substrate, adhesion to the microfiber was increased. (a) Low-density adhesion on the microfibers resulted in the axonal projections to the substrate. (b) Robust neuronal adhesion resulted in neuronal somata and axonal containment on the microfiber. Scale bar = 200 µm. Adapted with permission from Ref. 9, IOP Publishing.

FIGURE 7. Neuronal encapsulation on microfibers. For future transplantation, removal from culture while maintaining neuronal network integrity and viability is necessary. In order to demonstrate this using hydrogel encapsulation, neurons were plated on collagen-coated conductive polymer microfibers and, at 6–9 days in vitro, encapsulated using 0.5–1.0% agarose. (a-c) Representative fluorescent confocal reconstructions of encapsulated neuronal cultures on microfibers stained to discriminate live cells (green) from the nuclei of dead cells (red) (scale bar = 200 µm). (a) The encapsulation process did not reduce the cell density or the cell viability versus nonencapsulated controls. Increased magnification of regions of interest showing (b) a cluster of neuronal somata and (c) a neurite-rich segment following encapsulation (scale bars = 50 µm). Adapted with permission from Ref. 9, IOP Publishing. Volume 39, Number 3, 2011

256 Cullen et al.

to utilize tissue engineered constructs consisting of living neural cells within 3-D matrices, which may enhance regeneration or neural interface following neural injury or disease. Prior to in vivo delivery, these constructs may be pre-engineered in vitro with defined functional, geometric, and neuroanatomical features based on application, and may possess anisotropic features or a homogenous cell distribution, with or without electrical interface technology. For neuroregeneration, we have developed tissue engineered constructs to mitigate axonal loss, a prominent feature following trauma or disease in the nervous system. Our overall strategy is to create 3-D living axonal tracts that are designed to recapitulate lost neuroanatomy (Fig. 8). In one case, we mechanically engineer constructs consisting of long axonal tracts—up to 10 cm in length—which we have applied successfully to facilitate neuroregeneration following severe peripheral nerve or spinal cord injury.63,65 This technique is based on the ability of integrated axons to respond to continuous mechanical tension by exhibiting stretchgrowth, which produces progressively longer axons that gradually coalesce into large nerve tracts.59,60,62 In another approach, we engineered 3-D tubular microconduits with internalized longitudinally aligned, living axonal tracts. Due to their micronscale size, these microconduits may be delivered via injection in a minimally invasive fashion. In future applications, these 3-D microconduits may be precisely delivered to potentially reconnect neuronal populations following axonal loss. In addition, we have observed substantial axonal growth longitudinally along conductive microfibers, creating another technique for directed axonal extension. Based on these techniques and perhaps others, the transplantation of living axons pre-engineered to recapitulate lost axonal tracts may restore function by directly replacing lost connections or by serving as a scaffold to promote targeted host regeneration. For the latter purpose, the axonal constructs may create “regenerative highways” for targeted nerve tract restoration by exploiting axon-mediated axon regeneration. Our other application of neural tissue en-

gineering technology is to biohybridize neuralelectrical relays for functional integration with the host nervous system. This biohybridization of neural interface technology through a blending with neural tissue engineering techniques may directly promote host axonal integration, and therefore chronic communication. Accordingly, we are developing biohybridized neural interface microsystems consisting of living tissue engineered neural cellular constructs encapsulated within electrically active microarrays. These biohybridized microsystems may augment neuroregenerative capabilities in vivo and may be efficacious in improving axonal integration due to biologically mediated pathfinding. In addition, in the case of loss of limb, these biohybridized microsystems may serve as a surrogate end target, and thus provide a bidirectional electrically active neural interface to relay motor commands from and sensory signals to the spinal cord and brain. The long-term goal of these efforts is to create a robust, direct interface with the nervous system to drive sophisticated prosthetic devices for sustained man-machine interface. There are tremendous challenges for the in vivo survival and efficacy of tissue engineered neural constructs and/or biohybrid interface platforms. In addition to functional characteristics, issues pertaining to inflammation/immunogenicity and mass transport are paramount. Some degree of construct optimization may occur in vitro; however, challenges regarding survival and function following in vivo delivery will be inevitable. As strategies are developed to mitigate these challenges, the ability to engineer these nerve constructs with tailored cellular, anatomical, geometric, and functional characteristics will be extremely beneficial. With regard to immune tolerance, it may be possible to modulate construct attributes to modify the immunogenicity of the transplanted cells. This issue will be influenced by the goal of the construct. For instance, axonal conduits serving as regenerative scaffolds need remain only until host axons have reinnervated a target. Conversely, if the goal is to permanently replace lost connections, then chronic immune tolerance is required. We have found surprising survival, and hence imCritical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

257

FIGURE 8. Techniques to achieve longitudinal axonal extension and/or growth. Confocal reconstructions of neuronal constructs achieved via (a) stretch-growth, (b) microconduit containment, or (c) along a microfiber (each at 6–9 days in vitro). (a) Axon stretch-growth results in two neuronal populations spanned by long axonal tracts. (b) A dense cluster of neuronal somata was located at one end of the microconduits with axonal projections extending longitudinally, projecting several millimeters in the interior (outer diameter denoted by dashed lines). (c) Preferential longitudinal growth along a conductive polymer microfiber. (c) Adapted with permission from Ref. 9, IOP Publishing.

mune tolerance, of neuron-only constructs, which may be due to a low immunogenicity in these cells or rapid integration with the host nervous system. Other strategies such as cell/genetic engineering may further decrease the immunogenicity without compromising function. Also, attenuation of the foreign-body reaction to any biomaterial/microelectrodes may require systemic or local delivery of anti-inflammatory agents. In addition, unintended functionality will also need to be evaluated to avoid aberrant connectivity and thus achieve desired functional restoration. Advances in neuroregeneration could profoundly impact millions of patients suffering from brain injuries, spinal cord injuries, or neurodegenerative disorders. Here, neural tissue engineering offers tremendous therapeutic promise, and thus tissue engineered constructs to restore lost neurological function are aggressively being pursued. In addition, chronic neuroprosthetic interfaces providing seamless motor and sensory control Volume 39, Number 3, 2011

would greatly improve the quality of life of thousands dealing with loss of limb amongst other debilitating peripheral nerve injuries. Moreover, the biohybridization of neural interface technology through a blending with neural tissue engineering techniques may promote functional integration with the host nervous system. In these areas, the successful application involves the integration of engineered living tissue with the host nervous system to mitigate deficits, directly restore lost function, or to augment the capacity for nervous system regeneration. Acknowledgments This work was partially supported by the Department of Defense, the National Institutes of Health, and the National Science Foundation. The authors thank Drs. Niranjan Kameswaran, Min TangSchomer, Victoria E. Johnson, Kevin D. Browne, and Ankur R. Patel for technical contributions.

258 Cullen et al.

REFERENCES

1. Fu SY, Gordon T. Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy. J Neurosci. 1995;15(5 Pt 2):3876–85. 2. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol. 1997;14(1–2):67–116. 3. Gordon T, Chan KM, Sulaiman OA, Udina E, Amirjani N, Brushart TM. Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury. Neurosurgery. 2009;65(4 Suppl):A132–44. 4. Mackinnon SE, Dellon AL. A study of nerve regeneration across synthetic (Maxon) and biologic (collagen) nerve conduits for nerve gaps up to 5 cm in the primate. J Reconstr Microsurg. 1990;6(2):117–21. 5. Moore AM, Kasukurthi R, Magill CK, Farhadi HF, Borschel GH, Mackinnon SE. Limitations of conduits in peripheral nerve repairs. Hand (N Y). 2009;4(2):180–6. 6. Bunge MB. Bridging areas of injury in the spinal cord. Neuroscientist. 2001;7(4):325–39. 7. Houle JD, Tessler A. Repair of chronic spinal cord injury. Exp Neurol. 2003;182(2):247–60. 8. Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and regeneration. Annu Rev Biomed Eng. 2003;5:293–347. 9. Cullen DK, Patel A, Doorish JF, Smith DH, Pfister BJ. Developing a tissue engineered neuralelectrical relay using encapsulated neuronal constructs on conducting polymer fibers. J Neural Eng. 2008;5(4):374–84. 10. Kameswaran N, Cullen DK, Pfister BJ, Ranalli NJ, Huang JH, Zager EL, Smith DH. A novel neuroprosthetic interface with the peripheral nervous system using artificially engineered axonal tracts. Neurol Res. 2008c;30(10):1063–7. 11. Bellamkonda RV. Peripheral nerve regeneration: an opinion on channels, scaffolds and anisotropy. Biomaterials. 2006;27(19):3515–8. 12. Gilbert RJ, Rivet CJ, Zuidema JM, Popovich PG. Biomaterial design considerations for repairing the injured spinal cord. Crit Rev Biomed Eng. 2011;39(1). 13. Fawcett J. Repair of spinal cord injuries: where are we, where are we going? Spinal Cord. 2002;40(12):615–23.

14. Gordon T. The physiology of neural injury and regeneration: The role of neurotrophic factors. J Commun Disord. 2010;43(4):265–73. 15. Nisbet DR, Crompton KE, Horne MK, Finkelstein DI, Forsythe JS. Neural tissue engineering of the CNS using hydrogels. J Biomed Mater Res B Appl Biomater. 2008;87(1):251–63. 16. McKerracher L. Spinal cord repair: strategies to promote axon regeneration. Neurobiol Dis. 2001;8(1):11–8. 17. Evans GR. Peripheral nerve injury: a review and approach to tissue engineered constructs. Anat Rec. 2001;263(4):396–404. 18. Huang JH, Zager EL, Zhang J, Groff RF, Pfister BJ, Cohen AS, Grady MS, Maloney-Wilensky E, Smith DH. Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation. Laboratory investigation. J Neurosurg. 2008;108(2):343–7. 19. Hudson TW, Evans GR, Schmidt CE. Engineering strategies for peripheral nerve repair. Orthop Clin North Am. 2000;31(3):485–98. 20. Tallantyre EC, Bo L, Al-Rawashdeh O, Owens T, Polman CH, Lowe JS, Evangelou N. Clinicopathological evidence that axonal loss underlies disability in progressive multiple sclerosis. Mult Scler. 2010;16(4):406–11. 21. Belal A, Ylikoski J. Pathology as it relates to ear surgery II. Labyrinthectomy. J Laryngol Otol. 1983;97(1):1–10. 22. Levin PS, Newman SA, Quigley HA, Miller NR. A clinicopathologic study of optic neuropathies associated with intracranial mass lesions with quantification of remaining axons. Am J Ophthalmol. 1983;95(3):295–306. 23. Cheng HC, Ulane CM, Burke RE. Clinical progression in Parkinson disease and the neurobiology of axons. Ann Neurol. 2010;67(6):715–25. 24. Marshall VG, Bradley WG, Jr., Marshall CE, Bhoopat T, Rhodes RH. Deep white matter infarction: correlation of MR imaging and histopathologic findings.Radiology.1988;167(2):517– 22. 25. Siebert JR, Middelton FA, Stelzner DJ. Intrinsic response of thoracic propriospinal neurons to axotomy. BMC Neurosci. 2010;11:69. 26. Kelley BJ, Farkas O, Lifshitz J, Povlishock JT. Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotoCritical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

my and Wallerian degeneration. Exp Neurol. 2006;198(2):350–60. 27. Chew DJ, Leinster VH, Sakthithasan M, Robson LG, Carlstedt T, Shortland PJ. Cell death after dorsal root injury. Neurosci Lett. 2008;433(3):231–4. 28. Yip HK, Rich KM, Lampe PA, Johnson EM, Jr. The effects of nerve growth factor and its antiserum on the postnatal development and survival after injury of sensory neurons in rat dorsal root ganglia. J Neurosci. 1984;4(12):2986–92. 29. Curinga G, Smith GM. Molecular/genetic manipulation of extrinsic axon guidance factors for CNS repair and regeneration. Exp Neurol. 2008;209(2):333–42. 30. Huebner EA, Strittmatter SM. Axon regeneration in the peripheral and central nervous systems. Results Probl Cell Differ. 2009;48:339–51. 31. Hall S. Nerve repair: a neurobiologist’s view. J Hand Surg [Br]. 2001;26(2):129–36. 32. Jain A, Brady-Kalnay SM, Bellamkonda RV. Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension. J Neurosci Res. 2004;77(2):299–307. 33. Liu K, Lu Y, Lee JK, Samara R, Willenberg R, Sears-Kraxberger I, Tedeschi A, Park KK, Jin D, Cai B, et al. PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci. 2010;13(9):1075–81. 34. Yip PK, Wong LF, Sears TA, Yanez-Munoz RJ, McMahon SB. Cortical overexpression of neuronal calcium sensor-1 induces functional plasticity in spinal cord following unilateral pyramidal tract injury in rat. PLoS Biol. 2010;8(6):e1000399. 35. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon SB. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature. 2002;416(6881):636–40. 36. Stichel CC, Hermanns S, Luhmann HJ, Lausberg F, Niermann H, D’Urso D, Servos G, Hartwig HG, Müller HW. Inhibition of collagen IV deposition promotes regeneration of injured CNS axons. Eur J Neurosci. 1999;11(2):632–46. 37. Mingorance A, Sole M, Muneton V, Martinez A, Nieto-Sampedro M, Soriano E, del Río JA. Regeneration of lesioned entorhino-hippocampal Volume 39, Number 3, 2011

259

axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo66/NgR signaling. FASEB J. 2006;20(3):491–3. 38. Moore MJ, Friedman JA, Lewellyn EB, Mantila SM, Krych AJ, Ameenuddin S, Knight AM, Lu L, Currier BL, Spinner RJ, et al. Multiple-channel scaffolds to promote spinal cord axon regeneration. Biomaterials. 2006;27(3):419–29. 39. Silva NA, Salgado AJ, Sousa RA, Oliveira JT, Pedro AJ, Leite-Almeida H, Mastronardi F, Mano JF, et al. Development and characterization of a novel hybrid tissue engineering-based scaffold for spinal cord injury repair. Tissue Eng Part A. 2010;16(1):45–54. 40. Tsai EC, Dalton PD, Shoichet MS, Tator CH. Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection. J Neurotrauma. 2004;21(6):789–804. 41. Houle JD, Tom VJ, Mayes D, Wagoner G, Phillips N, Silver J. Combining an autologous peripheral nervous system “bridge” and matrix modification by chondroitinase allows robust, functional regeneration beyond a hemisection lesion of the adult rat spinal cord. J Neurosci. 2006;26(28):7405–15. 42. Wen X, Tresco PA. Effect of filament diameter and extracellular matrix molecule precoating on neurite outgrowth and Schwann cell behavior on multifilament entubulation bridging device in vitro. J Biomed Mater Res A. 2006;76(3):626–37. 43. Wen X, Tresco PA. Fabrication and characterization of permeable degradable poly(DL-lactideco-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels. Biomaterials. 2006;27(20):3800–9. 44. Whitlock EL, Tuffaha SH, Luciano JP, Yan Y, Hunter DA, Magill CK, et al. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve. 2009;39(6):787–99. 45. Cai J, Peng X, Nelson KD, Eberhart R, Smith GM. Permeable guidance channels containing microfilament scaffolds enhance axon growth and maturation. J Biomed Mater Res A. 2005;75(2):374–86. 46. Kim YT, Haftel VK, Kumar S, Bellamkonda RV. The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve

260 Cullen et al.

gaps. Biomaterials. 2008;29(21):3117–27. 47. Dodla MC, Bellamkonda RV. Anisotropic scaffolds facilitate enhanced neurite extension in vitro. J Biomed Mater Res A. 2006;78(2):213–21. 48. Dodla MC, Bellamkonda RV. Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps. Biomaterials. 2008;29(1):33–46. 49. Bozkurt A, Brook GA, Moellers S, Lassner F, Sellhaus B, Weis J, Woeltje M, Tank J, Beckmann C, Fuchs P, et al. In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix. Tissue Eng. 2007;13(12):2971–9. 50. Kim SM, Lee SK, Lee JH. Peripheral nerve regeneration using a three dimensionally cultured schwann cell conduit. J Craniofac Surg. 2007;18(3):475–88. 51. Tang XQ, Heron P, Mashburn C, Smith GM. Targeting sensory axon regeneration in adult spinal cord. J Neurosci. 2007;27(22):6068–78. 52. Orlacchio A, Bernardi G, Martino S. Stem cells: an overview of the current status of therapies for central and peripheral nervous system diseases. Curr Med Chem. 2010;17(7):595–608. 53. Kim HJ. Stem cell potential in Parkinson’s disease and molecular factors for the generation of dopamine neurons. Biochim Biophys Acta. 2010;1812(1):1–11. 54. Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, Summers R, Gage FH, Anderson AJ. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A. 2005;102(39):14069–74. 55. Mayer E, Fawcett JW, Dunnett SB. Basic fibroblast growth factor promotes the survival of embryonic ventral mesencephalic dopaminergic neurons—II. Effects on nigral transplants in vivo. Neuroscience. 1993;56(2):389–98. 56. Tate MC, Shear DA, Hoffman SW, Stein DG, Archer DR, LaPlaca MC. Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain. Cell Transplant. 2002;11(3):283–95. 57. Burnett MG, Zager EL. Pathophysiology of peripheral nerve injury: a brief review. Neurosurg Focus. 2004;16(5):E1.

58. East E, de Oliveira DB, Golding JP, Phillips JB. Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit. Tissue Eng Part A. 2010;16(10):3173–84. 59. Smith DH. Stretch growth of integrated axon tracts: extremes and exploitations. Prog Neurobiol. 2009;89(3):231–9. 60. Pfister BJ, Iwata A, Meaney DF, Smith DH. Extreme stretch growth of integrated axons. J Neurosci. 2004;24(36):7978–83. 61. Pfister BJ, Iwata A, Taylor AG, Wolf JA, Meaney DF, Smith DH. Development of transplantable nervous tissue constructs comprised of stretch-grown axons. J Neurosci Methods. 2006;153(1):95–103. 62. Smith DH, Wolf JA, Meaney DF. A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension. Tissue Eng. 2001;7(2):131–9. 63. Iwata A, Browne KD, Pfister BJ, Gruner JA, Smith DH. Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions. Tissue Eng. 2006n;12(1):101–10. 64. Pfister BJ, Bonislawski DP, Smith DH, Cohen AS. Stretch-grown axons retain the ability to transmit active electrical signals. FEBS Lett. 2006;580(14):3525–31. 65. Huang JH, Cullen DK, Browne KD, Groff R, Zhang J, Pfister BJ, et al. Long-term survival and integration of transplanted engineered nervous tissue constructs promotes peripheral nerve regeneration. Tissue Eng Part A. 2009;15(7):1677–85. 66. Cullen DK, Tang-Schomer MD, Johnson VE, Patel AR, Browne KD, Smith DH, editors. Tissue engineered microconduits for targeted restoration of axonal tracts. Biomedical Engineering Society Annual Meeting; 2010; Austin, TX. 67. Donoghue JP. Connecting cortex to machines: recent advances in brain interfaces. Nat Neurosci. 2002;5 Suppl:1085–8. 68. Hochberg LR, Serruya MD, Friehs GM, Mukand JA, Saleh M, Caplan AH, Branner A, Chen D, Penn RD, Donoghue JP. Neuronal ensemble control of prosthetic devices by a human with tetraplegia. Nature (London). Critical Reviews™ in Biomedical Engineering

Neural Tissue Engineering and Biohybridized Microsystems In Vivo

2006;442(7099):164–71. 69. Kuiken TA, Dumanian GA, Lipschutz RD, Miller LA, Stubblefield KA. The use of targeted muscle reinnervation for improved myoelectric prosthesis control in a bilateral shoulder disarticulation amputee. Prosthet Orthot Int. 2004;28(3):245–53. 70. Lebedev MA, Nicolelis MA. Brain-machine interfaces: past, present and future. Trends Neurosci. 2006;29(9):536–46. 71. Navarro X, Krueger TB, Lago N, Micera S, Stieglitz T, Dario P. A critical review of interfaces with the peripheral nervous system for the control of neuroprostheses and hybrid bionic systems. J Peripher Nerv Syst. 2005;10(3):229–58. 72. Taylor DM, Tillery SI, Schwartz AB. Direct cortical control of 3D neuroprosthetic devices. Science. 2002;296(5574):1829–32. 73. Wolpaw JR, Birbaumer N, McFarland DJ, Pfurtscheller G, Vaughan TM. Brain-computer interfaces for communication and control. Clin Neurophysiol. 2002;113(6):767–91. 74. Lee H, Bellamkonda RV, Sun W, Levenston ME. Biomechanical analysis of silicon microelectrode-induced strain in the brain. J Neural Eng. 2005;2(4):81–9.

Volume 39, Number 3, 2011

261

75. McConnell GC, Schneider TM, Owens DJ, Bellamkonda RV. Extraction force and cortical tissue reaction of silicon microelectrode arrays implanted in the rat brain. IEEE Trans Biomed Eng. 2007;54(6 Pt 1):1097–107. 76. Patrick E, Ordonez M, Alba N, Sanchez JC, Nishida T. Design and fabrication of a flexible substrate microelectrode array for brain machine interfaces. Conf Proc IEEE Eng Med Biol Soc. 2006;1:2966–9. 77. Pfister BJ, Huang JH, Kameswaran N, Zager EL, Smith DH. Neural engineering to produce in vitro nerve constructs and neurointerface. Neurosurgery. 2007;60(1):137–41; discussion 41–2. 78. Merrill DR, Tresco PA. Impedance characterization of microarray recording electrodes in vitro. IEEE Trans Biomed Eng. 2005;52(11):1960–5. 79. Wong JY, Langer R, Ingber DE. Electrically conducting polymers can noninvasively control the shape and growth of mammalian cells. Proc Natl Acad Sci U S A. 1994;91(8):3201–4. 80. Richardson-Burns SM, Hendricks JL, Martin DC. Electrochemical polymerization of conducting polymers in living neural tissue. J Neural Eng. 2007;4(2):L6–L13.

TISSUE ENGINEERING IN NERVOUS SYSTEM NOTES 2.pdf ...

of tissue engineered 3-D neural constructs may significantly advance regeneration or device-based deficit mitiga- tion in the nervous system that has not been achieved by non–tissue engineering approaches. KEY WORDS: neural engineering, neuroengineering, 3-D neural culture, axon, neuron, peripheral nerve injury,.

9MB Sizes 2 Downloads 142 Views

Recommend Documents

nervous system
നോഡീോകോശം - ഘടന. Page 12. TYPICAL NERVE CELL. Page 13. Page 14. Page 15. Page 16. TRANSMISSION OF IMPULSES. Page 17. ആോവഗപസരണം ...

anatomy of nervous system pdf
Sign in. Loading… Whoops! There was a problem loading more pages. Retrying... Whoops! There was a problem previewing this document. Retrying.

The-Central-Nervous-System-In-AIDS-Neurology-Radiology ...
Try one of the apps below to open or edit this item. The-Central-Nervous-System-In-AIDS-Neurology-Radiology-Pathology-Ophthalmology.pdf.

autonomic nervous system pharmacology pdf
Try one of the apps below to open or edit this item. autonomic nervous system pharmacology pdf. autonomic nervous system pharmacology pdf. Open. Extract.

OCT-In-Central-Nervous-System-Diseases-The-Eye ...
intracranial hypertension, Friedreich's ataxia, schizophrenia, hereditary optic neuropathies, glaucoma, and amblyopia. Individual chapters are also ... pharmacological treatment, and the use of OCT in animal models. ... total on the internet computer

pdf-0741\3d-bioprinting-and-nanotechnology-in-tissue-engineering ...
... apps below to open or edit this item. pdf-0741\3d-bioprinting-and-nanotechnology-in-tissue-e ... icine-by-lijie-grace-zhang-john-p-fisher-kam-leong.pdf.

pdf-0741\3d-bioprinting-and-nanotechnology-in-tissue-engineering ...
... apps below to open or edit this item. pdf-0741\3d-bioprinting-and-nanotechnology-in-tissue-e ... icine-by-lijie-grace-zhang-john-p-fisher-kam-leong.pdf.

Regenerative-Dentistry-Synthesis-Lectures-On-Tissue-Engineering ...
Retrying... Whoops! There was a problem previewing this document. Retrying... Download. Connect more apps... Try one of the apps below to open or edit this item. Regenerative-Dentistry-Synthesis-Lectures-On-Tissue-Engineering.pdf. Regenerative-Dentis

[PDF BOOK] Tissue Engineering
... CAncer CAD CAM acronym for Computer Aided Design Computer Aided Manufacturer or ... mellitus IDDM We Nanoscience and nanotechnology are transforming materials science in ... Read Best Book Online Tissue Engineering: Engineering Principles for the

Divisions of the Nervous System - notes.pdf
Axons can be as long as ______ in length or very short. Page 3 of 5. Divisions of the Nervous System - notes.pdf. Divisions of the Nervous System - notes.pdf.

anatomy of central nervous system pdf
pdf. Download now. Click here if your download doesn't start automatically. Page 1 of 1. anatomy of central nervous system pdf. anatomy of central nervous ...

pdf-1836\fine-structure-of-the-nervous-system-neurons ...
... the apps below to open or edit this item. pdf-1836\fine-structure-of-the-nervous-system-neurons- ... s-by-alan-peters-sanford-l-palay-henry-def-webster.pdf.

Work plan for the Central Nervous System Working Party 2017
Dec 15, 2016 - Guideline on the clinical development of medicinal products for the ... The guideline was developed in collaboration with BSWP, PDCO and ...

The Nervous System Independently Controls Motion ...
evidence for the existence of independent neural controllers for arm motion and ... 1Desmurget et al., Nature Neuroscience 2(6), 1999; Della-Maggiore et al., ...

Engineering Notes
In recent years, a number of research works have been published .... be defined as a gradient of a virtual potential function U as follows: ..... 800. Relative Position. Time (hour). Distance (m) x y z. Fig. 2 Rendezvous maneuver in a circular orbit