The traditional notion that injured neurons are unable to regenerate in the adult mammalian brain and spinal cord has long been a concern. This view has led to methodology designed to overcome this problem, most recently by advancements in tissue engineering. Here, neural precursor cells (NPCs) and the Nogo receptor antibody (NgR-Ab) or poly-L-lysine (PLL) were tested in concert with hyaluronic acid hydrogel scaffolds (HA). In particular, we wished to optimize viability and differentiation of NPCs within HA hydrogel scaffolds. Our results show that HA hydrogels can be modified physically or chemically to improve NPCs attachment on the scaffolding doped with NgR-Ab or PLL. Both the HA hydrogels and their modifications support the viability of NPCs. NPCs were also able to differentiate into neurons and glial cells on HA hydrogels, although this was affected by the different modifications. Immunofluorescence showed that fewer beta-III-tubulin antibody and antineurofilament antibody-positive cells were found on HA-PLL hydrogel compared with HA or HA NgR-Ab hydrogels. This indicates that the PLL-modified HA hydrogels may inhibit differentiation of NPCs, whereas modification by NgR-Ab had no such effect. Finally, the NgR-Ab-modified HA scaffold can be used as not only a NPC delivery system but also a bioactive factor transportation system for CNS repair.

We report extraordinary perpendicular orientations of neurons dependent on the presence of an external direct current (DC) voltage gradient. We chose chick dorsal root and postganglionic sympathetic neurons to evaluate. These were cultured in observation chambers in which the cells were separated from electrode products or substrate effects and maintained at 35°C. Both types of neurons showed a rapid restructuring of their anatomy. Typically, neurites that were not perpendicular to the voltage gradient were quickly resorbed into the cell body within a few minutes. Over 3-6 hr, significant new neurite growth occurred and was patterned perpendicular to the DC electrical field (Ef). This preferred asymmetry was dependent on the Ef, as was the initial retrograde degeneration of fibers. At 400-500 mV/mm, over 90% of the cells in culture assumed this orientation. Removal of the DC Ef led to a loss of the preferred orientation, with further random growth within the chambers. This is the first report of such responses in dorsal root ganglion neurons. We also used sympathetic neurons as a meaningful comparison to analyze whether there were any qualitative or quantitative differences between these two cell types of neural crest origin. We discuss the means by which these orientations were achieved.

Direct current (DC) electrical stimulation has been shown to have remarkable effects on regulating cell behaviors. Translation of this technology to clinical uses, however, has to overcome several obstacles, including Joule heat production, changes in pH and ion concentration, and electrode products that are detrimental to cells. Application of DC voltages in thick tissues where their thickness is >0.8 mm caused significant changes in temperature, pH, and ion concentrations. In this study, we developed a multifield and -chamber electrotaxis chip, and various stimulation schemes to determine effective and safe stimulation strategies to guide the migration of human vascular endothelial cells. The electrotaxis chip with a chamber thickness of 1 mm allows 10 voltages applied in one experiment. DC electric fields caused detrimental effects on cells in a 1 mm chamber that mimicking 3D tissue with a decrease in cell migration speed and an increase in necrosis and apoptosis. Using the chip, we were able to select optimal stimulation schemes that were effective in guiding cells with minimal detrimental effects. This experimental system can be used to determine optimal electrical stimulation schemes for cell migration, survival with minimal detrimental effects on cells, which will facilitate to bring electrical stimulation for in vivo use.

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The remarkable polarity-dependent growth and anatomical organization of neurons in vitro produced by imposed direct current (DC) voltage gradients (electrical fields; Ef) can be mimicked by another type of electrical cue. This is a properly structured asymmetrical alternating current (AC) electrical field (A-ACEf). Here we provide details on the construction of an AC signal generator in which all components of an AC waveform can be individually controlled. We show that 1) conventional symmetrical AC voltage gradients will not induce growth, guidance, or architectural changes in sympathetic neurons. We also provide the first qualitative and quantitative data showing that an asymmetric AC application can indeed mimic the DC response in chick sympathetic neurons and their growing neurites. This shift in orientation and neuronal anatomy requires dieback of some neurites and the extension of others to produce a preferred orientation perpendicular to the gradient of voltage. Our new results may lead to a noninvasive means to modify nerve growth and organization by magnetic inductive coupling at distance. These data also indicate the possibility of a means to mimic DC-dependent release of drugs or other biologically active molecules from electrically sensitive that can be loaded with these chemical cargos.