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In JoVE (1)
Other Publications (16)
- Annals of Biomedical Engineering
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Journal of Neuroscience Methods
- Tissue Engineering
- FEBS Letters
- Neurosurgery
- Progress in Brain Research
- Journal of Neurosurgery
- Journal of Neural Engineering
- Neurological Research
- Tissue Engineering. Part A
- The American Journal of Forensic Medicine and Pathology
- Critical Reviews in Biomedical Engineering
- Journal of Neurotrauma
- Journal of Neurotrauma
- Critical Reviews in Biomedical Engineering
Articles by Bryan J. Pfister in JoVE
JoVE Bioengineering
Axon Stretch Growth: The Mechanotransduction of Neuronal Growth
1Departments of Biomedical Engineering, New Jersey Institute of Technology, 2Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey
A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.
Other articles by Bryan J. Pfister on PubMed
An in Vitro Uniaxial Stretch Model for Axonal Injury
We have developed a unique uniaxial stretching device to study axonal injury and neural cell death resulting from brain tissue deformations common in traumatic head injuries. Using displacement control rather than force control, this device is capable of achieving strains >70% and strain rates up to 90 s(-1), well above those currently used for studying axonal injury. We have demonstrated that the deformation of the specimen was uniaxial, uniform and highly reproducible; the prespecified displacement profiles could be realized almost precisely; and adequate cell adhesion could be achieved readily. The entire device can fit into a biological safety cabinet to maintain sterility, and the specimens are convenient for cell culture. This device can be used to investigate a wide range of biomechanical issues involved in diffuse axonal injury.
Extreme Stretch Growth of Integrated Axons
Large animals can undergo enormous growth during development, suggesting that axons in nerves and white matter tracts rapidly expand as well. Because integrated axons have no growth cones to extend from, it has been postulated that mechanical forces may stimulate axon elongation matching the growth of the animal. However, this distinct form of rapid and sustained growth of integrated axons has never been demonstrated. Here, we used a microstepper motor system to evaluate the effects of escalating rates of stretch on integrated axon tracts over days to weeks in culture. We found that axon tracts could be stretch grown at rates of 8 mm/d and reach lengths of 10 cm without disconnection. Despite dynamic and long-term elongation, stretched axons increased in caliber by 35%, while the morphology and density of cytoskeletal constituents and organelles were maintained. These data provide the first evidence that mechanical stimuli can induce extreme "stretch growth" of integrated axon tracts, far exceeding any previously observed limits of axon growth.
Development of Transplantable Nervous Tissue Constructs Comprised of Stretch-grown Axons
Pursuing a new approach to nervous system repair, fasciculated axon tracts grown in vitro were developed into nervous tissue constructs designed to span peripheral nerve or spinal cord lesions. We optimized the newfound process of extreme axon stretch growth to maximize the number and length of axon tracts, reach an unprecedented axon growth-rate of 1cm/day, and create 5cm long axon tracts in 8 days to serve as the core component of a living nervous tissue construct. Immunocytochemical analysis confirmed that elongating fibers were axons, and that all major cytoskeletal constituents were present across the stretch-growth regions. We formed a transplantable nervous tissue construct by encasing the elongated cells in an 80% collagen hydrogel, removing them from culture, and inserting them into a synthetic conduit. Alternatively, we induced axon stretch growth directly on a surgical membrane that could be removed from the elongation device, and formed into a cylindrical construct suitable for transplant. The ability to rapidly create living nervous tissue constructs that recapitulates the uniaxial orientations of the original nerve offers an unexplored and potentially complimentary direction in nerve repair. Ideally, bridging nerve damage with living axon tracts may serve to establish or promote new functional connections.
Long-term Survival and Outgrowth of Mechanically Engineered Nervous Tissue Constructs Implanted into Spinal Cord Lesions
While most approaches to repair spinal cord injury (SCI) rely on promoting axon outgrowth, the extensive distance that axons would have to grow to bridge SCI lesions remains an enormous challenge. In this study, we used a new tissue-engineering technique to create long nervous tissue constructs spanned by living axon tracts to repair long SCI lesions. Exploiting the newfound process of extreme axon stretch growth, integrated axon tracts from dorsal root ganglia (DRG) neurons were mechanically elongated in vitro to 10 mm over 7 days and encased in a collagen hydrogel to form a nervous tissue construct. In addition, a modified lateral hemisection SCI model in the rat was developed to create a 1 cm long cavity in the spinal cord. Ten days following SCI, constructs were transplanted into the lesion and the animals were euthanized 4 weeks post-transplantation for histological analyses. Through cell tracking methods and immunohistochemistry, the transplanted elongated cultures were consistently found to survive 4 weeks in the injured spinal cord. In addition, DRG axons were observed extending out of the transplanted construct into the host spinal cord tissue. These results demonstrate the promise of nervous tissue constructs consisting of stretch-grown axons to bridge even extensive spinal cord lesions.
Stretch-grown Axons Retain the Ability to Transmit Active Electrical Signals
Little is known about extensive nervous system growth after axons reach their targets. Indeed, postnatal animals continue to grow, suggesting that axons are stretched to accommodate the expanding body. We have previously shown that axons can sustain stretch-growth rates reaching 1 cm/day; however, it remained unknown whether the ability to transmit active signals was maintained. Here, stretch-growth did not alter sodium channel activation, inactivation, and recovery or potassium channel activation. In addition, neurons generated normal action potentials that propagated across stretch-grown axons. Surprisingly, Na and K channel density increased due to stretch-growth, which may represent a natural response to preserve the fidelity of neuronal signaling.
Neural Engineering to Produce in Vitro Nerve Constructs and Neurointerface
Recently, our laboratory recapitulated a natural form of axon growth that occurs between late embryogenesis and early adulthood. In this article, we describe how this novel neural engineering approach may be used to produce a nervous tissue interface to integrate disconnected motor and sensory functions for external control.
Injury-induced Alterations in CNS Electrophysiology
Mild to moderate cases of traumatic brain injury (TBI) are very common, but are not always associated with the overt pathophysiogical changes seen following severe trauma. While neuronal death has been considered to be a major factor, the pervasive memory, cognitive and motor function deficits suffered by many mild TBI patients do not always correlate with cell loss. Therefore, we assert that functional impairment may result from alterations in surviving neurons. Current research has begun to explore CNS synaptic circuits after traumatic injury. Here we review significant findings made using in vivo and in vitro models of TBI that provide mechanistic insight into injury-induced alterations in synaptic electrophysiology. In the hippocampus, research now suggests that TBI regionally alters the delicate balance between excitatory and inhibitory neurotransmission in surviving neurons, disrupting the normal functioning of synaptic circuits. In another approach, a simplified model of neuronal stretch injury in vitro, has been used to directly explore how injury impacts the physiology and cell biology of neurons in the absence of alterations in blood flow, blood brain barrier integrity, or oxygenation associated with in vivo models of brain injury. This chapter discusses how these two models alter excitatory and inhibitory synaptic transmission at the receptor, cellular and circuit levels and how these alterations contribute to cognitive impairment and a reduction in seizure threshold associated with human concussive brain injury.
Harvested Human Neurons Engineered As Live Nervous Tissue Constructs: Implications for Transplantation. Laboratory Investigation
Although neuron transplantation to repair the nervous system has shown promise in animal models, there are few practical sources of viable neurons for clinical application and insufficient approaches to bridge extensive nerve damage in patients. Therefore, the authors sought a clinically relevant source of neurons that could be engineered into transplantable nervous tissue constructs. The authors chose to evaluate human dorsal root ganglion (DRG) neurons due to their robustness in culture.
Developing a Tissue-engineered Neural-electrical Relay Using Encapsulated Neuronal Constructs on Conducting Polymer Fibers
Neural-electrical interface platforms are being developed to extracellularly monitor neuronal population activity. Polyaniline-based electrically conducting polymer fibers are attractive substrates for sustained functional interfaces with neurons due to their flexibility, tailored geometry and controlled electro-conductive properties. In this study, we addressed the neurobiological considerations of utilizing small diameter (<400 microm) fibers consisting of a blend of electrically conductive polyaniline and polypropylene (PA-PP) as the backbone of encapsulated tissue-engineered neural-electrical relays. We devised new approaches to promote survival, adhesion and neurite outgrowth of primary dorsal root ganglion neurons on PA-PP fibers. We attained a greater than ten-fold increase in the density of viable neurons on fiber surfaces to approximately 700 neurons mm(-2) by manipulating surrounding surface charges to bias settling neuronal suspensions toward fibers coated with cell-adhesive ligands. 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 PA-PP 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. These efforts accomplished key prerequisites for the establishment of functional electrical interfaces with neuronal populations using small diameter PA-PP fibers-specifically, improved neurocompatibility, high-density neuronal adhesion and neuritic network development directly on fiber surfaces.
A Novel Neuroprosthetic Interface with the Peripheral Nervous System Using Artificially Engineered Axonal Tracts
We have previously described a technique developed in our laboratory to create transplantable living axon tracts of several centimeters in length. In this paper, we describe how these engineered neural tissue constructs can be used to create a novel neuroelectrical interface with the regenerating peripheral nervous system, to potentially enable afferent and efferent communications with prosthetic devices.
Long-term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration
Although peripheral nerve injury is a common consequence of trauma or surgery, there are insufficient means for repair. In particular, there is a critical need for improved methods to facilitate regeneration of axons across major nerve lesions. Here, we engineered transplantable living nervous tissue constructs to provide a labeled pathway to guide host axonal regeneration. These constructs consisted of stretch-grown, longitudinally aligned living axonal tracts inserted into poly(glycolic acid) tubes. The constructs (allogenic) were transplanted to bridge an excised segment of sciatic nerve in the rat, and histological analyses were performed at 6 and 16 weeks posttransplantation to determine graft survival, integration, and host regeneration. At both time points, the transplanted constructs were found to have maintained their pretransplant geometry, with surviving clusters of graft neuronal somata at the extremities of the constructs spanned by tracts of axons. 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. By 16 weeks posttransplant, extensive myelination of axons was observed throughout the transplant region. Further, graft neurons had extended axons beyond the margins of the transplanted region, penetrating into the host nerve. Notably, this survival and integration of the allogenic constructs occurred in the absence of immunosuppression therapy. 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.
Head Motions While Riding Roller Coasters: Implications for Brain Injury
The risk of traumatic brain injury (TBI) while riding roller coasters has received substantial attention. Case reports of TBI around the time of riding roller coasters have led many medical professionals to assert that the high gravitational forces (G-forces) induced by roller coasters pose a significant TBI risk. Head injury research, however, has shown that G-forces alone cannot predict TBI. Established head injury criterions and procedures were employed to compare the potential of TBI between daily activities and roller coaster riding. Three-dimensional head motions were measured during 3 different roller coaster rides, a pillow fight, and car crash simulations. Data was analyzed and compared with published data, using similar analyses of head motions. An 8.05 m/s car crash lead to the largest head injury criterion measure of 28.1 and head impact power of 3.41, over 6 times larger than the roller coaster rides of 4.1 and 0.36. Notably, the linear and rotational components of head acceleration during roller coaster rides were milder than those induced by many common activities. As such, there appears to be an extremely low risk of TBI due to the head motions induced by roller coaster rides.
Biomedical Engineering Strategies for Peripheral Nerve Repair: Surgical Applications, State of the Art, and Future Challenges
Damage to the peripheral nervous system is surprisingly common and occurs primarily from trauma or a complication of surgery. Although recovery of nerve function occurs in many mild injuries, outcomes are often unsatisfactory following severe trauma. Nerve repair and regeneration presents unique clinical challenges and opportunities, and substantial contributions can be made through the informed application of biomedical engineering strategies. This article reviews the clinical presentations and classification of nerve injuries, in addition to the state of the art for surgical decision-making and repair strategies. This discussion presents specific challenges that must be addressed to realistically improve the treatment of nerve injuries and promote widespread recovery. In particular, nerve defects a few centimeters in length use a sensory nerve autograft as the standard technique; however, this approach is limited by the availability of donor nerve and comorbidity associated with additional surgery. Moreover, we currently have an inadequate ability to noninvasively assess the degree of nerve injury and to track axonal regeneration. As a result, wait-and-see surgical decisions can lead to undesirable and less successful "delayed" repair procedures. In this fight for time, degeneration of the distal nerve support structure and target progresses, ultimately blunting complete functional recovery. Thus, the most pressing challenges in peripheral nerve repair include the development of tissue-engineered nerve grafts that match or exceed the performance of autografts, the ability to noninvasively assess nerve damage and track axonal regeneration, and approaches to maintain the efficacy of the distal pathway and targets during the regenerative process. Biomedical engineering strategies can address these issues to substantially contribute at both the basic and applied levels, improving surgical management and functional recovery following severe peripheral nerve injury.
Live Imaging of Axon Stretch Growth in Embryonic and Adult Neurons
Strategies for nervous system repair arise from knowledge of growth mechanisms via a growth cone. The distinctive process of axon stretch growth is a robust, long-term growth that may reveal new pathways to accelerate nerve repair. Here, a live imaging bioreactor was engineered to closely explore cellular events initiated by applied tension. The stretch growth potential between adult and embryonic dorsal root ganglion (DRG) neurons was investigated, an important difference in nerve repair. Embryonic axons were capable of unidirectional stretch growth rates of 4?mm/d and reliably reached 4?cm in length within 2 weeks. Adult axons could only reach 2?mm/d and took over 3 weeks to reach 4?cm. Utilizing time-lapse imaging, we observed growth cone motility in coordination with stretch growth. Upon initiation of stretching, growth cones retracted. However, within 10?h of continuous stretching, growth cones extended at a rate of 0.2?mm/d opposite the direction of applied tension, contributing to overall axon elongation. We analyzed fast mitochondrial transport under increasing levels of strain to determine the effect of stretch on axonal transport. Transport began to diminish at 24% strain, and was almost completely absent at 39% strain. Surprisingly, axons recovered and were capable of subsequent stretch growth. When tension was completely released (?5% strain), stretch grown axons retracted at rates up to 6.1??m/sec and slowed as resting tension was restored. This ability to assess the process of axon stretch growth in real time will allow detailed study of how tension can be used to drive axonal growth and retraction.
Engineering a High Throughput Axon Injury System
Several key biological mechanisms of traumatic injury to axons have been elucidated using in vitro stretch injury models. These models, however, are based on the experimentation of single cultures keeping productivity slow. Indeed, low yield has hindered important and well-founded investigations requiring high throughput methods such as proteomic analyses. To meet this need, we engineered a multi-well high throughput injury device to accelerate and accommodate the next generation of traumatic brain injury research. This modular system stretch injures neuronal cultures in either a 24-well culture plate format or 6 individual wells simultaneously. Custom software control allows the user to accurately program the pressure pulse parameters to achieve the desired substrate deformation and injury parameters. Analysis of the pressure waveforms showed that peak pressure was linearly related to input pressure and valve open times and that the 6- and 24-well modules displayed rise times, peak pressures, and decays with extremely small standard deviations. Data also confirmed that the pressure pulse was distributed evenly throughout the pressure chambers and therefore to each injury well. Importantly, the relationship between substrate deformation and applied pressure was consistent among the multiple wells and displayed a predictable linear behavior in each module. These data confirm that this multi-well system performs as well as currently used stretch injury devices and can undertake high throughput studies that are needed across the field of neurotrauma research.
Neural Tissue Engineering for Neuroregeneration and Biohybridized Interface Microsystems in Vivo (Part 2)
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.
