The Effect of Electrospun Fiber Diameter on Astrocyte-Mediated Neurite Guidance and Protection.

The topography of electrospun fiber scaffolds modifies astrocytes toward in vivo-like morphologies and behaviors. However, little is known about how electrospun fiber diameter influences astrocyte behavior. In this work, aligned fibers with two distinct nanoscale fiber diameters (808 and 386 nm) were prepared, and the astrocyte response was measured over time. Astrocytes on the large diameter fibers showed significantly increased elongation as early as 2 h after seeding and remained significantly more elongated for up to 4 days compared to those on small diameter fibers. Astrocytes extending along larger diameter fibers were better equipped to support long neurite outgrowth from dorsal root ganglia neurons, and neurite outgrowth along these astrocytes was less branched than outgrowth along astrocytes cultured on small diameter fibers. The differences in astrocyte shape observed on the small or large diameter fibers did not translate into differences in GLT-1, GFAP, or GLAST protein expression. Thus, different fiber diameters were unable to influence astrocyte protein expression uniquely. Nevertheless, astrocytes cultured in either small or large fibers significantly increased their expression of GLT-1 compared to astrocytes cultured on nonfiber (film) controls. Fibrous-induced increases in astrocyte GLT-1 expression protected astrocyte/neuron cocultures from toxicity generated by high extracellular glutamate. Alternatively, astrocytes/neurons cultured on films were less able to protect these cells from culture conditions consisting of high glutamate levels. Biomaterials, such as the fibrous materials presented here, may help stimulate astrocytes to increase GLT-1 expression and uptake more glutamate, since astrocytes are less likely to uptake glutamate in neurodegenerative pathologies or following central nervous system injury.

Injectable, Magnetically Orienting Electrospun Fiber Conduits for Neuron Guidance.

Magnetic electrospun fibers are of interest for minimally invasive biomaterial applications that also strive to provide cell guidance. Magnetic electrospun fibers can be injected and then magnetically positioned in situ, and the aligned fiber scaffolds provide consistent topographical guidance to cells. In this study, magnetically responsive aligned poly-l-lactic acid electrospun fiber scaffolds were developed and tested for neural applications. Incorporating oleic acid-coated iron oxide nanoparticles significantly increased neurite outgrowth, reduced the fiber alignment, and increased the surface nanotopography of the electrospun fibers. After verifying neuron viability on two-dimensional scaffolds, the system was tested as an injectable three-dimensional scaffold. Small conduits of aligned magnetic fibers were easily injected in a collagen or fibrinogen hydrogel solution and repositioned using an external magnetic field. The aligned magnetic fibers provided internal directional guidance to neurites within a three-dimensional collagen or fibrin model hydrogel, supplemented with Matrigel. Neurites growing from dorsal root ganglion explants extended 1.4-3× farther on the aligned fibers compared with neurites extending in the hydrogel alone. Overall, these results show that magnetic electrospun fiber scaffolds can be injected and manipulated with a magnetic field in situ to provide directional guidance to neurons inside an injectable hydrogel. Most importantly, this injectable guidance system increased both neurite alignment and neurite length within the hydrogel scaffold.

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage.

The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures. Endothelial factors generated under dynamic conditions significantly increased neuronal differentiation, while those released under static conditions stimulated oligodendrocyte differentiation. Flow increases EC release of neurogenic factors and of heparin sulfate glycosaminoglycans that increase their bioactivity, likely underlying the enhanced neuronal differentiation. Additionally, endothelial factors, especially from static conditions, promoted adherent growth. Together, our data suggest that blood flow may impact proliferation, adhesion, and the neuron-glial fate choice of adult NPCs, with implications for diseases and aging that reduce flow.

Nebulized solvent ablation of aligned PLLA fibers for the study of neurite response to anisotropic-to-isotropic fiber/film transition (AFFT) boundaries in astrocyte-neuron co-cultures.

Developing robust in vitro models of in vivo environments has the potential to reduce costs and bring new therapies from the bench top to the clinic more efficiently. This study aimed to develop a biomaterial platform capable of modeling isotropic-to-anisotropic cellular transitions observed in vivo, specifically focusing on changes in cellular organization following spinal cord injury. In order to accomplish this goal, nebulized solvent patterning of aligned, electrospun poly-l-lactic acid (PLLA) fiber substrates was developed. This method produced a clear topographic transitional boundary between aligned PLLA fibers and an isotropic PLLA film region. Astrocytes were then seeded on these scaffolds, and a shift between oriented and non-oriented astrocytes was created at the anisotropic-to-isotropic fiber/film transition (AFFT) boundary. Orientation of chondroitin sulfate proteoglycans (CSPGs) and fibronectin produced by these astrocytes was analyzed, and it was found that astrocytes growing on the aligned fibers produced aligned arrays of CSPGs and fibronectin, while astrocytes growing on the isotropic film region produced randomly-oriented CSPG and fibronectin arrays. Neurite extension from rat dissociated dorsal root ganglia (DRG) was studied on astrocytes cultured on anisotropic, aligned fibers, isotropic films, or from fibers to films. It was found that neurite extension was oriented and longer on PLLA fibers compared to PLLA films. When dissociated DRG were cultured on the astrocytes near the AFFT boundary, neurites showed directed orientation that was lost upon growth into the isotropic film region. The AFFT boundary also restricted neurite extension, limiting the extension of neurites once they grew from the fibers and into the isotropic film region. This study reveals the importance of anisotropic-to-isotropic transitions restricting neurite outgrowth by itself. Furthermore, we present this scaffold as an alternative culture system to analyze neurite response to cellular boundaries created following spinal cord injury and suggest its usefulness to study cellular responses to any aligned-to-unorganized cellular boundaries seen in vivo.

Electrical stimuli in the central nervous system microenvironment.

Electrical stimulation to manipulate the central nervous system (CNS) has been applied as early as the 1750s to produce visual sensations of light. Deep brain stimulation (DBS), cochlear implants, visual prosthetics, and functional electrical stimulation (FES) are being applied in the clinic to treat a wide array of neurological diseases, disorders, and injuries. This review describes the history of electrical stimulation of the CNS microenvironment; recent advances in electrical stimulation of the CNS, including DBS to treat essential tremor, Parkinson's disease, and depression; FES for the treatment of spinal cord injuries; and alternative electrical devices to restore vision and hearing via neuroprosthetics (retinal and cochlear implants). It also discusses the role of electrical cues during development and following injury and, importantly, manipulation of these endogenous cues to support regeneration of neural tissue.

Rapid assessment of migration and proliferation: a novel 3D high-throughput platform for rational and combinatorial screening of tissue-specific biomaterials.

Designing an ideal biomaterial supportive of multicellular tissue repair is challenging, especially with a poor understanding of the synergy between constituent proteins and growth factors. A brute-force approach, based on screening all possible combinations of proteins and growth factors, is inadequate due to the prohibitively large experimental space coupled with current low-throughput screening techniques. A high-throughput screening platform based on rational and combinatorial strategies for design and testing of proteins and growth factors can significantly impact the discovery of novel tissue-specific biomaterials. Here, we report the development of a flexible high-throughput screening platform, Rapid Assessment of Migration and Proliferation (RAMP), to rapidly investigate cell viability, proliferation, and migration in response to highly miniaturized three-dimensional biomaterial cultures (4-20 µL) with sparingly low cell densities (63-1000 cells per µL for cell arrays; 1 µL of 1000-10,000 cells per µL for migration arrays). The predictions made by RAMP on the efficacy and potency of the biomaterials are in agreement with the predictions made by conventional assays but at a throughput that is at least 100-1000-fold higher. The RAMP assay is therefore a novel approach for the rapid discovery of tissue-specific biomaterials for tissue engineering and regenerative medicine.

Electrical stimulation of schwann cells promotes sustained increases in neurite outgrowth.

Endogenous electric fields are instructive during embryogenesis by acting to direct cell migration, and postnatally, they can promote axonal growth after injury (McCaig 1991, Al-Majed 2000). However, the mechanisms for these changes are not well understood. Application of an appropriate electrical stimulus may increase the rate and success of nerve repair by directly promoting axonal growth. Previously, DC electrical stimulation at 50 mV/mm (1 mA, 8 h duration) was shown to promote neurite outgrowth and a more pronounced effect was observed if both peripheral glia (Schwann cells) and neurons were co-stimulated. If electrical stimulation is delivered to an injury site, both the neurons and all resident non-neuronal cells [e.g., Schwann cells, endothelial cells, fibroblasts] will be treated and this biophysical stimuli can influence axonal growth directly or indirectly via changes to the resident, non-neuronal cells. In this work, non-neuronal cells were electrically stimulated, and changes in morphology and neuro-supportive cells were evaluated. Schwann cell response (morphology and orientation) was examined after an 8 h stimulation over a range of DC fields (0-200 mV/mm, DC 1 mA), and changes in orientation were observed. Electrically prestimulating Schwann cells (50 mV/mm) promoted 30% more neurite outgrowth relative to co-stimulating both Schwann cells with neurons, suggesting that electrical stimulation modifies Schwann cell phenotype. Conditioned medium from the electrically prestimulated Schwann cells promoted a 20% increase in total neurite outgrowth and was sustained for 72 h poststimulation. An 11-fold increase in nerve growth factor but not brain-derived neurotrophic factor or glial-derived growth factor was found in the electrically prestimulated Schwann cell-conditioned medium. No significant changes in fibroblast or endothelial morphology and neuro-supportive behavior were observed poststimulation. Electrical stimulation is widely used in clinical settings; however, the rational application of this cue may directly impact and enhance neuro-supportive behavior, improving nerve repair.

Neurite outgrowth is significantly increased by the simultaneous presentation of Schwann cells and moderate exogenous electric fields.

Axonal extension is influenced by a variety of external guidance cues; therefore, the development and optimization of a multi-faceted approach is probably necessary to address the intricacy of functional regeneration following nerve injury. In this study, primary dissociated neonatal rat dorsal root ganglia neurons and Schwann cells were examined in response to an 8 h dc electrical stimulation (0-100 mV mm(-1)). Stimulated samples were then fixed immediately, immunostained, imaged and analyzed to determine Schwann cell orientation and characterize neurite outgrowth relative to electric field strength and direction. Results indicate that Schwann cells are viable following electrical stimulation with 10-100 mV mm(-1), and retain a normal morphology relative to unstimulated cells; however, no directional bias is observed. Neurite outgrowth was significantly enhanced by twofold following exposure to either a 50 mV mm(-1) electric field (EF) or co-culture with unstimulated Schwann cells by comparison to neurons cultured alone. Neurite outgrowth was further increased in the presence of simultaneously applied cues (Schwann cells + 50 mV mm(-1) dc EF), exhibiting a 3.2-fold increase over unstimulated control neurons, and a 1.2-fold increase over either neurons cultured with unstimulated Schwann cells or the electrical stimulus alone. These results indicate that dc electric stimulation in combination with Schwann cells may provide synergistic guidance cues for improved axonal growth relevant to nerve injuries in the peripheral nervous system.

Generation of cell-derived three dimensional extracellular matrix substrates from two dimensional endothelial cell cultures.

Within the cellular microenvironment, extracellular matrix (ECM) proteins are critical nonsoluble signaling factors that modulate cell attachment, migration, proliferation, and differentiation. We have developed a simple method to isolate and process ECM from endothelial cell cultures to create a three-dimensional (3D) ECM substrate. Endothelial cell monolayers were chemically lysed and enzymatically digested to isolate a thin, two-dimensional (2D) ECM substrate. This thin 1.8 µm 2D ECM was collected and applied to a solid support to produce 12-16-fold thicker 3D ECM substrates with average thicknesses ranging from 21 to 29 µm. The biological activity of isolated ECM was assessed by cell culture. Neural progenitor cells were cultured on endothelial-produced ECM, and unlike the thin 2D ECM, which was quickly remodeled by cells, 3D ECM substrates remained in culture for an extended period (>7 days), suggesting that a continuous signaling cue for in vitro experiments may be provided. This simple method for creating 3D ECM substrates can be applied to a variety of cell culture models for studies aimed at identifying the signaling effects of the ECM within cellular microenvironments.

Single-walled carbon nanotubes alter Schwann cell behavior differentially within 2D and 3D environments.

Both spinal cord injury (SCI) and large-gap peripheral nerve defects can be debilitating affecting a patient's long-term quality of life and presently, there is no suitable treatment for functional regeneration of these injured tissues. A number of works have suggested the benefits of electrical stimulation to promote both glial migration and neuronal extension. In this work, an electrically conductive hydrogel containing single-walled carbon nanotubes (SWCNT) for neural engineering applications is presented and the Schwann cell (SC) response to SWCNT is examined in both 2D and 3D microenvironments. Results from clonogenic and alamarBlue® assays in 2D indicate that SWCNT (10-50 µg mL(-1)) inhibit SC proliferation but do not affect cell viability. Following SWCNT exposure in 2D, changes in SC morphology can be observed with the nanomaterial attached to the cell membrane at concentrations as low as 10 µg mL(-1). In contrast to the results gathered in 2D, SC embedded within the 3D hydrogel loaded with 10-50 µg mL(-1) of SWCNT exhibited little or no measurable change in cell proliferation, viability, or morphology as assessed using a digestion assay, alamarBlue, and confocal microscopy. Collectively, this highlights that an electrically-conductive SWCNT collagen I-Matrigel™ biomaterial may be suitable for neural tissue engineering and is able to sustain populations of SC. Findings suggest that 2D nanoparticle toxicity assays may not be accurate predictors of the 3D response, further motivating the examination of these materials in a more physiologically relevant environment.

Collagen I-matrigel scaffolds for enhanced Schwann cell survival and control of three-dimensional cell morphology.

We report on the ability to control three-dimensional Schwann cell (SC) morphology using collagen I-Matrigel composite scaffolds for neural engineering applications. SCs are supportive of nerve regeneration after injury, and it has recently been reported that SCs embedded in collagen I, a material frequently used in guidance channel studies, do not readily extend processes, instead adopting a spherical morphology indicative of little interaction with the matrix. We have modified collagen I matrices by adding Matrigel to make them more supportive of SCs and characterized these matrices and SC morphology in vitro. Incorporation of 10%, 20%, 35%, and 50% Matrigel by volume resulted in 2.4, 3.5, 3.7, and 4.2 times longer average SC process length after 14 days in culture than with collagen I-only controls. Additionally, only 35% and 50% Matrigel constructs were able to maintain SC number over 14 days, whereas an 88% decrease in cells from initial seeding density was observed in collagen-only constructs over the same time period. Mechanical testing revealed that the addition of 50% Matrigel increased matrix stiffness from 6.4 kPa in collagen I-only constructs to 9.8 kPa. Furthermore, second harmonic generation imaging showed that the addition of Matrigel resulted in non-uniform distribution of collagen I, and scanning electron microscope imaging illustrated distinct differences in the fibrillar structure of the different constructs. Collectively, this work lays a foundation for developing scaffolding materials that are concurrently supportive of neurons and SCs for future neural engineering applications.

Cryopreservation of transfected primary dorsal root ganglia neurons.

Primary dorsal root ganglia (DRG) neurons are often used to investigate the relative strength of various guidance cues to promote re-growth in vitro. Current methods of neuron isolation are laborious and disposal of excess dissected cells is inefficient. Traditional immunostaining techniques are inadequate to visualize real-time neurite outgrowth in co-culture. Cryopreservation, in combination with transfection techniques, may provide a viable solution to both under-utilized tissue and insufficient methods of visualization. This study aims to qualitatively and quantitatively demonstrate successful cryopreservation of primary transfected and non-transfected DRG neurons. Fluorescent micrographs were used to assess morphology after 24h in culture and suggest similarities between freshly isolated neurons and neurons which have been transfected and/or cryopreserved. Quantitative measurements of neuron outgrowth (specifically, primary neurites, branch points and total neurite length) indicate that neuron outgrowth is not altered by cryopreservation. Transfected neurons have stunted outgrowth at 24h.

Impact of carbon nanotube exposure, dosage and aggregation on smooth muscle cells.

Carbon nanotubes (CNT), despite their diverse application potential, have demonstrated adverse impacts in vitro and in vivo. Previous studies have focused on the combined in vitro cytotoxic impact of CNT aggregates and associated nanoparticulate impurities. However, the isolated effect of CNT aggregates and associated non-aggregated nanoparticulates have not been addressed in detail. In this work, the impact of single-walled nanotubes (SWNT) on rat aortic smooth muscle cells (SMC) was examined for SWNT (0.0-0.1 mg/ml) over a 3.5-day time-course. Cell culture medium was filtered to remove the aggregate material and both nanomaterial (un-filtered) and filtered SWNT media were used to examine cell growth. In general, the removal of SWNT aggregates from cell culture test medium by filtration increased the SMC number in comparison to unfiltered medium at pre-filtered SWNT dosages below 0.1 mg/ml. However, at 0.1 mg/ml, both filtered and unfiltered media exhibited a similar decrease in cell number relative to the control medium. The filtered medium was characterized and contained both suspended nanoparticles as well as a small quantity of SWNT, which may have contributed to the observed cell growth inhibition. As a comparison to the SWNT, activated carbon (0.1 mg/ml), a nanoporous, microparticulate carbon material, was found to be less inhibitory to SMC growth than the SWNT at the same dosage, implying an inverse proportionality between carbon nanomaterial size regimes and cell growth inhibition.

Neurite outgrowth is directed by schwann cell alignment in the absence of other guidance cues.

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micro-patterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal-Schwann cell interactions in vitro.

Optimization of reporter cells for expression profiling in a microfluidic device.

The emergence of green fluorescence protein (GFP) technologies has enabled non-invasive monitoring of cell function and gene expression. GFP-based expression studies are typically performed in traditional single-dish or multi-well formats to monitor a small number of genes or conditions that do not lend well to scaling, high-throughput analysis, or single-cell measurements. We have recently developed a microfluidic device, the Living Cell Array (LCA), for monitoring GFP-based gene expression in a high-throughput manner. Here, we report the optimization of GFP reporter cell characteristics in this microfluidic device for gene expression profiling. A reporter cell line for the transcription factor NF-kappa B was generated and used as the model cell line. Reporter cells were seeded in the LCA and NF-kappa B activated by addition of the cytokine TNF-alpha . Our studies show that the fluorescence kinetics from the reporter cell line in response to both single and repeated TNF-alpha stimulation in the LCA is similar to that observed in standard tissue culture. In addition, our data also indicate that multiple expression waves can be reliably monitored from a small population of reporter cells. Using reporter cell line subcloning and cell cycle synchronization, we demonstrate that the kinetics and magnitude of induced fluorescence in the reporter cell lines can be further improved to maximize the fluorescence readout from reporter cell lines, thereby improving their applicability to live cell expression profiling. Our studies establish some of the important criteria to be considered when using reporter cell lines for dynamic expression profiling in microfluidic devices.

Oriented Schwann cell monolayers for directed neurite outgrowth.

Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration. They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface. In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance. Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D. M., and H. M. Buettner. Tissue Eng. 7(3):247-266, 2001). In the current study, these substrates were used to create oriented Schwann cell monolayers. Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions. In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment. In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium. This produced complete coverage without large seeding densities while preserving alignment to the micropattern. Alignment and coverage were unaffected by micropattern dimensions. This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.

Dynamic gene expression profiling using a microfabricated living cell array.

We describe the development of a microfluidic platform for continuous monitoring of gene expression in live cells. This optically transparent microfluidic device integrates high-throughput molecular stimulation with nondestructive monitoring of expression events in individual living cells, hence, a living cell array (LCA). Several concentrations of a soluble molecular stimulus are generated in an upstream microfluidic network and used to stimulate downstream reporter cells, each containing a green fluorescence reporter plasmid for a gene of interest. Cellular fluorescence is continuously monitored and quantified to infer the expression dynamics of the gene being studied. We demonstrate this approach by profiling the activation of the transcription factor NF-kappaB in HeLa S3 cells in response to varying doses of the inflammatory cytokine TNF-alpha. The LCA platform offers a unique opportunity to simultaneously control dynamic inputs and measure dynamic outputs from adherent mammalian cells in a high-throughput fashion. This approach to profiling expression dynamics, in conjunction with complementary techniques such as DNA microarrays, will help provide a more complete picture of the dynamic cellular response to diverse soluble stimuli.