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.

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.

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.

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 DRG 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 (PLLA) 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. 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 neurite, indicating topographical cues are responsible to guide 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.

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.