Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

University of Connecticut Health Center & Connecticut Children's Medical Center

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This work describes a method to bond patterned macromolecular gels into monolithic structures using perturbants. Bonding strengths for a variety of solutes follow a Hofmeister ordering; this result and optical measurements indicate that bonding occurs by reversible perturbation of contacting gels. The resulting microfluidic gels are mechanically robust and can serve as scaffolds for cell culture.

Bonding of macromolecular hydrogels using perturbants.

Ce travail d&eacute;crit une m&eacute;thode pour lier des gels macromol&eacute;culaires &agrave; motifs dans des structures monolithiques &agrave; l&#39;aide de perturbateurs. Les forces de liaison pour une vari&eacute;t&eacute; de solut&eacute;s suivent un classement Hofmeister ; ce r&eacute;sultat et des mesures optiques indiquent que la liaison se fait par perturbation r&eacute;versible de communiquer avec les gels. Les gels de microfluidique qui en r&eacute;sultent sont m&eacute;caniquement robustes et peuvent servir d&#39;&eacute;chafaudages pour la culture cellulaire.

Collage des hydrogels macromol&eacute;culaires &agrave; l&#39;aide de perturbateurs.

Diese Arbeit beschreibt ein Verfahren zur gemusterten Makromolekulare Gele in monolithischer Strukturen mit Perturbants zu verbinden. Bindung st&auml;rken f&uuml;r eine Vielzahl von Analyten folgen eine Sortierung der Hofmeister; Dieses Ergebnis und optische Messungen zeigen, dass die Bindung durch reversible St&ouml;rung der Kontaktaufnahme mit Gele auftritt. Die resultierende Microfluidic-Gele sind mechanisch robust und k&ouml;nnen als Ger&uuml;ste f&uuml;r Zellkultur dienen.

Verkleben von makromolekularen Hydrogele mit Perturbants.

Questo lavoro descrive un metodo per bond gel macromolecolare fantasia in strutture monolitiche utilizzando perturbants. Forza di legame per una variet&agrave; di soluti segue un ordinamento Hofmeister; Questo risultato e misurazioni ottiche indicano che legame si verifica da perturbazione reversibile di contatto con il gel. Il gel di microfluidica risultante sono meccanicamente robusto e pu&ograve; servire come ponteggi per la coltura cellulare.

Incollaggio di idrogel macromolecolare utilizzando perturbants.

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Este trabajo describe un m&eacute;todo para enlazar modelados macromoleculares geles en estructuras monol&iacute;ticas usando perturbants. Fuerzas de adhesi&oacute;n para una variedad de solutos seguir un orden Hofmeister; Este resultado y mediciones &oacute;pticas indican que la vinculaci&oacute;n se produce por perturbaci&oacute;n reversible de ponerse en contacto con geles. Los geles de microfluidos resultante son mec&aacute;nicamente robustos y pueden servir como andamios para el cultivo celular.

La vinculaci&oacute;n de hidrogeles macromoleculares mediante perturbants.

Little is known about which components of the axonal cytoskeleton might break during rapid mechanical deformation, such as occurs in traumatic brain injury. Here, we micropatterned neuronal cell cultures on silicone membranes to induce dynamic stretch exclusively of axon fascicles. After stretch, undulating distortions formed along the axons that gradually relaxed back to a straight orientation, demonstrating a delayed elastic response. Subsequently, swellings developed, leading to degeneration of almost all axons by 24 h. Stabilizing the microtubules with taxol maintained the undulating geometry after injury but greatly reduced axon degeneration. Conversely, destabilizing microtubules with nocodazole prevented undulations but greatly increased the rate of axon loss. Ultrastructural analyses of axons postinjury revealed immediate breakage and buckling of microtubules in axon undulations and progressive loss of microtubules. Collectively, these data suggest that dynamic stretch of axons induces direct mechanical failure at specific points along microtubules. This microtubule disorganization impedes normal relaxation of the axons, resulting in undulations. However, this physical damage also triggers progressive disassembly of the microtubules around the breakage points. While the disintegration of microtubules allows delayed recovery of the "normal" straight axon morphology, it comes at a great cost by interrupting axonal transport, leading to axonal swelling and degeneration.

Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration.

Traumatic axonal injury (TAI) is the most common and important pathology of traumatic brain injury (TBI). However, little is known about potential indirect effects of TAI on dendrites. In this study, we used a well-established in vitro model of axonal stretch injury to investigate TAI-induced changes in dendrite morphology. Axons bridging two separated rat cortical neuron populations plated on a deformable substrate were used to create a zone of isolated stretch injury to axons. Following injury, we observed the formation of dendritic alterations or beading along the dendrite shaft. Dendritic beading formed within minutes after stretch then subsided over time. Pharmacological experiments revealed a sodium-dependent mechanism, while removing extracellular calcium exacerbated TAI's effect on dendrites. In addition, blocking ionotropic glutamate receptors with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 prevented dendritic beading. These results demonstrate that axon mechanical injury directly affects dendrite morphology, highlighting an important bystander effect of TAI. The data also imply that TAI may alter dendrite structure and plasticity in vivo. An understanding of TAI's effect on dendrites is important since proper dendrite function is crucial for normal brain function and recovery after injury.

Dendritic alterations after dynamic axonal stretch injury in vitro.

Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as "varicosities." Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury.

Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.

As a common feature of many neurological diseases and injury, the loss of axon pathways can have devastating effects on function. Here, we demonstrate a new strategy to restore damaged axon pathways using transplantable miniature constructs consisting of living neurons and axonal tracts internalized within hydrogel tubes. These hydrogel microconduits were developed through an iterative process to support neuronal survival and directed axon growth. The design included hollow agarose tubes providing a relatively stiff outer casing to direct constrained unidirectional outgrowth of axons through a central soft collagen matrix, with overall dimensions of 250 μm inner diameter ×500 μm outer diameter and extending up to several centimeters. The outer casing was also designed to provide structural support of neuronal/axonal cultures during transplantation of the construct. Using neuron culture conditions optimized for the microconduits, dissociated dorsal root ganglia neurons were seeded in the collagen at one end of the conduits. Over the following week, high-resolution confocal microscopy demonstrated that the neurons survived and the somata remained in a tight cluster at the original seeding site. In addition, robust outgrowth of axons from the neurons was found, with axon fascicles constrained in a longitudinal projection along the internal collagen canal and extending over 5 mm in length. Notably, this general geometry recapitulates the anatomy of axon tracts. As such, these constructs may be useful to repair damaged axon projections by providing a transplantable bridge of living axons. Moreover, the small size of the construct permits follow-on studies of minimally invasive transplantation into potentially sensitive regions of the nervous system.

Microtissue engineered constructs with living axons for targeted nervous system reconstruction.

Peripheral nerve injury is a critical issue for patients with trauma. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm × 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 μm wide × 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 min each day for 7 days. Responses were compared with neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared with the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on days 5 and 7 compared with the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 min daily, in addition to surface patterning, of 3.5 μm wide × 500 nm deep grooves, offered control of nerve axon outgrowth and alignment.

Neural responses to electrical stimulation on patterned silk films.

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WITHDRAWN: Neural circuits with long-distance axon tracts for determining functional connectivity.

The cortical circuitry in the brain consists of structurally and functionally distinct neuronal assemblies with reciprocal axon connections. To generate cell culture-based systems that emulate axon tract systems of an in vivo neural network, we developed a living neural circuit consisting of compartmentalized neuronal populations connected by arrays of two millimeter-long axon tracts that are integrated on a planar multi-electrode array (MEA). The millimeter-scale node-to-node separation allows for pharmacological and electrophysiological manipulations to simultaneously target multiple neuronal populations. The results show controlled selectivity of dye absorption by neurons in different compartments. MEA-transmitted electrical stimulation of targeted neurons shows ∼46% increase of intracellular calcium levels with 20 Hz stimulation, but ∼22% decrease with 2k Hz stimulation. The unique feature of long distance axons promotes in vivo-like fasciculation. These axon tracts are determined to be inhibitory afferents by showing increased action potential firing of downstream node upon selective application of γ-aminobutyric acid (GABA) to the upstream node. Together, this model demonstrates integrated capabilities for assessing multiple endpoints including axon tract tracing, calcium influx, network architecture and activities. This system can be used as a multi-functional platform for studying axon tract-associated CNS disorders in vitro, such as diffuse axonal injury after brain trauma.

Neural circuits with long-distance axon tracts for determining functional connectivity.

Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits.

The behavior of neuronal cells on tendon-derived collagen sheets as potential substrates for nerve regeneration.

Tunable protein composites are important for constructing extracellular matrix mimics of human tissues with control of biochemical, structural, and mechanical properties. Molecular interaction mechanisms between silk fibroin protein and recombinant human tropoelastin, based on charge, are utilized to generate a new group of multifunctional protein alloys (mixtures of silk and tropoelastin) with different net charges. These new biomaterials are then utilized as a biomaterial platform to control neuron cell response. With a +38 net charge in water, tropoelastin molecules provide extraordinary elasticity and selective interactions with cell surface integrins. In contrast, negatively charged silk fibroin protein (net charge -36) provides remarkable toughness and stiffness with morphologic stability in material formats via autoclaving-induced beta-sheet crystal physical crosslinks. The combination of these properties in alloy format extends the versatility of both structural proteins, providing a new biomaterial platform. The alloys with weak positive charges (silk/tropoelastin mass ratio 75/25, net charge around +16) significantly improved the formation of neuronal networks and maintained cell viability of rat cortical neurons after 10 days in vitro. The data point to these protein alloys as an alternative to commonly used poly-L-lysine (PLL) coatings or other charged synthetic polymers, particularly with regard to the versatility of material formats (e.g., gels, sponges, films, fibers). The results also provide a practical example of physically designed protein materials with control of net charge to direct biological outcomes, in this case for neuronal tissue engineering.

Charge-Tunable Silk-Tropoelastin Protein Alloys That Control Neuron Cell Responses.

The brain remains one of the most important but least understood tissues in our body, in part because of its complexity as well as the limitations associated with in vivo studies. Although simpler tissues have yielded to the emerging tools for in vitro 3D tissue cultures, functional brain-like tissues have not. We report the construction of complex functional 3D brain-like cortical tissue, maintained for months in vitro, formed from primary cortical neurons in modular 3D compartmentalized architectures with electrophysiological function. We show that, on injury, this brain-like tissue responds in vitro with biochemical and electrophysiological outcomes that mimic observations in vivo. This modular 3D brain-like tissue is capable of real-time nondestructive assessments, offering previously unidentified directions for studies of brain homeostasis and injury.

Bioengineered functional brain-like cortical tissue.

Neural engineering provides promise for cell therapy by integrating the host brain with brain-machine-interface technologies in order to externally modulate functions. Long-term interfaces with the host brain remain a critical challenge due to insufficient graft cell survivability and loss of brain electrode sensitivity over time. Here, integrated neuron-electrode interfaces were developed on thin flexible and transparent silk films as brain implants. Mechanical properties and surface topography of silk films were optimized to promote cell survival and alignment of primary rat cortical cells. Compartmentalized cultures of living neural circuit and co-patterned electrode arrays were incorporated on the silk films with built-in wire connections. Electrical stimulation via electrodes embedded in the films activated surrounding neurons evoked calcium responses. In mice brains the silk film implants showed conformal contact capable of modulating host brain cells with minimal inflammatory response and stable indwelling for weeks. The approach of combining cell therapy and brain electrodes could provide sustained functional brain-machine interfaces with ex vivo control of neuron-electrode interface with spatial and temporal precision.

Film-based Implants for Supporting Neuron-Electrode Integrated Interfaces for The Brain.

Recombinant spider silks produced in transgenic goat milk were studied as cell culture matrices for neuronal growth. Major ampullate spidroin 1 (MaSp1) supported neuronal growth, axon extension and network connectivity, with cell morphology comparable to the gold standard poly-lysine. In addition, neurons growing on MaSp1 films had increased neural cell adhesion molecule (NCAM) expression at both mRNA and protein levels. The results indicate that MaSp1 films present useful surface charge and substrate stiffness to support the growth of primary rat cortical neurons. Moreover, a putative neuron-specific surface binding sequence GRGGL within MaSp1 may contribute to the biological regulation of neuron growth. These findings indicate that MaSp1 could regulate neuron growth through its physical and biological features. This dual regulation mode of MaSp1 could provide an alternative strategy for generating functional silk materials for neural tissue engineering.

Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks.

A bioengineered model of 3D brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, which resembles the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro and anchoring of the central collagen gel to avoid shrinkage, and enabling neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies and as a drug screening tool, and it could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 d, and the resulting tissues can be maintained in culture for several weeks.

Min Tang-Schomer

Department of Pediatrics

University of Connecticut Health Center & Connecticut Children's Medical Center

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Min Tang-Schomer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

University of Connecticut Health Center & Connecticut Children's Medical Center

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Department of Pediatrics