Christopher J. Rivet

A protocol for rheological characterization of hydrogels for tissue engineering strategies.

Hydrogels are studied extensively for many tissue engineering applications, and their mechanical properties influence both cellular and tissue compatibility. However, it is difficult to compare the mechanical properties of hydrogels between studies due to a lack of continuity between rheological protocols. This study outlines a straightforward protocol to accurately determine hydrogel equilibrium modulus and gelation time using a series of rheological tests. These protocols are applied to several hydrogel systems used within tissue engineering applications: agarose, collagen, fibrin, Matrigel™, and methylcellulose. The protocol is outlined in four steps: (1) Time sweep to determine the gelation time of the hydrogel. (2) Strain sweep to determine the linear-viscoelastic region of the hydrogel with respect to strain. (3) Frequency sweep to determine the linear equilibrium modulus plateau of the hydrogel. (4) Time sweep with values obtained from strain and frequency sweeps to accurately report the equilibrium moduli and gelation time. Finally, the rheological characterization protocol was evaluated using a composite Matrigel™-methylcellulose hydrogel blend whose mechanical properties were previously unknown. The protocol described herein provides a standardized approach for proper analysis of hydrogel rheological properties.

Altering Iron Oxide Nanoparticle Surface Properties Induce Cortical Neuron Cytotoxicity

Superparamagnetic iron oxide nanoparticles, with diameters in the range of a few tens of nanometers, display the ability to cross the blood-brain barrier and are envisioned as diagnostic and therapeutic tools in neuro-medicine. However, despite the numerous applications being explored, insufficient information is available on their potential toxic effect on neurons. While iron oxide has been shown to pose a decreased risk of toxicity, surface functionalization, often employed for targeted delivery, can significantly alter the biological response. This aspect is addressed in the present study, which investigates the response of primary cortical neurons to iron oxide nanoparticles with coatings frequently used in biomedical applications: aminosilane, dextran, and polydimethylamine. Prior to administering the particles to neuronal cultures, each particle type was thoroughly characterized to assess the (1) size of individual nanoparticles, (2) concentration of the particles in solution, and (3) agglomeration size and morphology. Culture results show that polydimethylamine functionalized nanoparticles induce cell death at all concentrations tested by swift and complete removal of the plasma membrane. Aminosilane coated particles affected metabolic activity only at higher concentrations while leaving the membrane intact, and dextran-coated nanoparticles partially altered viability at higher concentrations. These findings suggest that nanoparticle characterization and primary cell-based cytotoxicity evaluation should be completed prior to applying nanomaterials to the nervous system.

Neurite outgrowth on electrospun PLLA fibers is enhanced by exogenous electrical stimulation

OBJECTIVE Both electrical stimuli (endogenous and exogenous) and topographical cues are instructive to axonal extension. This report, for the first time, investigated the relative dominance of directional topographical guidance cues and directional electrical cues to enhance and/or direct primary neurite extension. We hypothesized the combination of electrical stimulation with electrospun fiber topography would induce longer neurite extension from dorsal root ganglia neurons than the presence of electrical stimulation or aligned topography alone. APPROACH To test the hypothesis, neurite outgrowth was examined on laminin-coated poly-L-lactide films or electrospun fibers (2 µm in diameter) in the presence or absence of electrical stimulation. Immunostained neurons were semi-automatically traced using Neurolucida software and morphology was evaluated. MAIN RESULTS Neurite extension increased 74% on the aligned fibers compared to film controls. Stimulation alone increased outgrowth by 32% on films or fibers relative to unstimulated film controls. The co-presentation of topographical (fibers) with biophysical (electrical stimulation) cues resulted in a synergistic 126% increase in outgrowth relative to unstimulated film controls. Field polarity had no influence on the directionality of neurites, indicating topographical cues are responsible for guiding neurite extension. SIGNIFICANCE Both cues (electrical stimulation and fiber geometry) are modular in nature and can be synergistically applied in conjunction with other common methods in regenerative medicine such as controlled release of growth factors to further influence axonal growth in vivo. The combined application of electrical and aligned fiber topographical guidance cues described herein, if translated in vivo, could provide a more supportive environment for directed and robust axonal regeneration following peripheral nerve injury.

Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels

UNLABELLED The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-μg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-μg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinsons, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

Cell infiltration into a 3D electrospun fiber and hydrogel hybrid scaffold implanted in the brain.

Tissue engineering scaffolds are often designed without appropriate consideration for the translational potential of the material. Solid scaffolds implanted into central nervous system (CNS) tissue to promote regeneration may require tissue resection to accommodate implantation. Or alternatively, the solid scaffold may be cut or shaped to better fit an irregular injury geometry, but some features of the augmented scaffold may fail to integreate with surrounding tissue reducing regeneration potential. To create a biomaterial able to completely fill the irregular geometry of CNS injury and yet still provide sufficient cell migratory cues, an injectable, hybrid scaffold was created to present the physical architecture of electrospun fibers in an agarose/methylcellulose hydrogel. When injected into the rat striatum, infiltrating macrophages/microglia and resident astrocytes are able to locate the fibers and utilize their cues for migration into the hybrid matrix. Thus, hydrogels containing electrospun fibers may be an appropriate platform to encourage regeneration of the injured brain.

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.

A biomaterial model of tumor stromal microenvironment promotes mesenchymal morphology but not epithelial to mesenchymal transition in epithelial cells.

The stromal tissue surrounding most carcinomas is comprised of an extracellular matrix densely packed with collagen-I fibers, which are often highly aligned in metastatic disease. Here we developed an in vitro model to test the effect of an aligned fibrous environment on cancer cell morphology and behavior, independent of collagen ligand presentation. We grew cells on a biomimetic surface of aligned electrospun poly-l-lactic acid (PLLA) fibers and then examined the effect of this environment on growth rate, morphology, cytoskeletal organization, biochemical and genetic markers of epithelial to mesenchymal transition (EMT), cell surface adhesion, and cell migration. We grew a phenotypically normal breast epithelial cell line (MCF10A) and an invasive breast cancer cell line (MDA-MB-231) on three different substrates: typical flat culture surface (glass or plastic), flat PLLA (glass coated with PLLA) or electrospun PLLA fibers. Cells of both types adopted a more mesenchymal morphology when grown on PLLA fibers, and this effect was exaggerated in the more metastatic-like MDA-MB-231 cells. However, neither cell type underwent the changes in gene expression indicative of EMT despite the changes in cell shape, nor did they exhibit the decreased adhesive strength or increased migration typical of metastatic cells. These results suggest that changes in cell morphology alone do not promote a more mesenchymal phenotype and consequently that the aligned fibrous environment surrounding epithelial cancers may not promote EMT solely through topographical cues.

Effect of magnetic nanoparticle heating on cortical neuron viability

Abstract Purpose: Superparamagnetic iron oxide nanoparticles are currently approved for use as an adjunctive treatment to glioblastoma multiforme radiotherapy. Radio frequency stimulation of the nanoparticles generates localised hyperthermia, which sensitises the tumour to the effects of radiotherapy. Clinical trials reported thus far are promising, with an increase in patient survival rate; however, what are left unaddressed are the implications of this technology on the surrounding healthy tissue. Methods and materials: Aminosilane-coated iron oxide nanoparticles suspended in culture medium were applied to chick embryonic cortical neuron cultures. Cultures were heated to 37 °C or 45 °C by an induction coil system for 2 h. The latter regime emulates the therapeutic conditions of the adjunctive therapy. Cellular viability and neurite retraction was quantified 24 h after exposure to the hyperthermic events. Results: The hyperthermic load inflicted little damage to the neuron cultures, as determined by calcein-AM, propidium iodide, and alamarBlue® assays. Fluorescence imaging was used to assess the extent of neurite retraction which was found to be negligible. Conclusions: Retention of chick, embryonic cortical neuron viability was confirmed under the thermal conditions produced by radiofrequency stimulation of iron oxide nanoparticles. While these results are not directly applicable to clinical applications of hyperthermia, the thermotolerance of chick embryonic cortical neurons is promising and calls for further studies employing human cultures of neurons and glial cells.