In the past decade, there has been significant research investigating selective neural stem cell (NSC) differentiation using scaffolds engineered from novel materials with unique 3D microstructures [1]. The goal is to synthesize a controlled, nontoxic microenvironment mimicking the physical and chemical stimuli found in the extracellular matrix (ECM) of mature cells. A promising candidate for nerve cell engineering is carbon, which is innately non-toxic to living cells, and has ideal conductive properties for enhancing charge transport. Additionally, carbon has many allotropes, each with unique properties shown to direct stem cell differentiation [4], and can be engineered into 3D structures with tunable surface properties and geometries mimicking the ECM.

A material that is both electrically conductive and structurally mimetic only addresses two of the criteria in tricking NSCs towards specific lineages and morphologies. The third challenge is simulating the mechanical stresses experienced by stem cells from dynamic changes in the developing microstructure of the ECM or the folding macrostructural tissue [5]. There are four main mechanical loads soft matter can experience: stretching/compression, bending, shear, and torsion. Typically, standalone carbon scaffolds are too brittle to withstand the mechanical stimuli for these types of experiments, requiring support structures or composite carbon-polymer matrices to enhance their elasticity.

To address these challenges, we engineered a standalone porous, 3D carbon material with ideal conductive properties for electrical stimulation and a microstructure that allows for mechanical stimulation.

To create a carbon material with the structure and properties of nanofiber mats, PAN polymer nanofibers, infused with carbon nanotubes, were deposited using controlled electrospinning. This material was then pyrolyzed to transform the polymer into carbon. The final carbon exhibited a uniquely graphitized structure, abundant in edge planes, which translates into its electrochemical kinetics. Owing to its tunable microstructure, the new pyrolytic carbon can also endure cycles of mechanical loading, required in mechanical stimulation of stem cells.

Additionally, high-resolution electron microscopy of the synthesized carbon revealed fragmented yet well-aligned lattice fringes. This characterization suggests a high ratio of atomic edge planes to basal planes in the carbon structure. Such a high concentration of edge plane atoms makes for a highly reactive carbon surface, which holds great promise for enhanced electrochemical performance [7]. This observation is particularly interesting in the context of carbon’s capability to sense organic chemicals, such as dopamine, a neurotransmitter that modulates critical functions of the central nervous system [6]. Detection of dopamine products during stem cell differentiation is crucial in identifying stem cell fate. Figure 1 demonstrates the enhanced performance of our material in detecting dopamine, compared with pure glassy carbon (1a) and pure PAN electrodes (1b). Our mechanically treated PAN-CNT showed significantly fast and reversible kinetics and exhibited clearly defined peaks distinguishing dopamine from interfering ascorbic and uric acid.

The compatibility of the carbon material as a stem cell scaffold was assessed by growing and differentiating neural stem cells on the material. The scaffold was first plasma treated and coated with laminin to increase hydrophilicity and help stem cells attach to the scaffold. NSCs harvested from the medial ganglionic eminence of 14.5 day old mouse embryos were subsequently grown and allowed to differentiate for a total of 5 days on the scaffold. Biocompatibility of NSCs on the carbon material was determined using a Live/Dead assay (Molecular Probes, Invitrogen), performed 1, 3, and 5 days following seeding. Additionally, the orientation of stem cells on the 3D structure of the scaffold was determined using SEM imaging of seeded NSCs. These results demonstrate our graphitic carbon is an ideal neural stem cell scaffold, capable of real time monitoring of cellular metabolites while directing the growth and differentiation of neural stem cells.

REFERENCES:

[1]Zorlutuna, P. et al., Adv. Mater. 24, 1782–1804 (2012).

[2] Lovat, V. et al., Nano Lett. 5, 1107–1110 (2005).

[3] Li, Ning, et al., Scientific reports 3 (2013).

[4] Kotov, Nicholas A., et al., Advanced Materials 21.40 (2009): 3970-4004.

[5] Arulmoli, J., Scientific reports, 5, (2015).

[6] Ganz, J., et al. Expert Rev. Neurother. 11, 1325–1339 (2011).

[7] Ghazinejad,M. , et. al., Scientific reports, Accepted Nov 12, 2017.

Figure 1 Caption: Electrocatalytic oxidation of Dopamine, Ascorbic Acid and Uric Acid. Cyclic voltammograms of a) glassy carbon, b) pure PAN electrode and c) treated CNT-PAN in 10 mM Ascorbic Acid, 1 mM Uric Acid and 1mM Dopamine in a pH 7.2 PBS buffer. d.) separate voltammograms were obtained of each electroactive species and the results were overlaid.