Artificial “Axons” from Optoelectronic Silicon for Studying the Role of Electrical Activity in Oligodendrocytes Governed Myelination

Myelin is a protective lipid that encircles nerve cells that significantly increases action potential’s propagation speed and efficiency. Myelin degeneration degrades neuronal function and is associated in many neurodegenerative diseases such as multiple sclerosis. In the central nervous system, myelination is governed by oligodendrocytes, and although electrical activity plays a major role in this process, the underlying mechanism is not well understood. Myelination was found to be promoted in electrically active nerve, while neuron-independent intracellular calcium transients within the OLs themselves were also found to affect this process. Interestingly, this question has been thus far approached by imaging spontaneous calcium transients and correlating them to myelination outcomes. However, electrically modulating the myelinating axons during this process may add another layer and help develop bioelectrical protocols to enhance and induce myelination.<br/>The gold standard myelination in vitro model is using neurons and OLs co-cultures, however, these co-cultures are difficult to control as neurons have their own activity adding biological “noise” into the system. Thus, neuron free myelination models using nano- and micro-fibers that mimic axonal morphology were developed. However, although this reductionist approach allows robust and high throughout tool for studding different component of the myelination process, they lack the ability to induce local electrical activity.<br/>In this study, we developed new bioinspired artificial ”axons” (AAs) from silicon mesh designed to imitate neuronal morphology. We then used our previously reported approach for developing in situ porosity-based optoelectronic heterojunction to make these AAs photo-responsive. We hypothesized that by optically inducing electrical activity in AAs interfacing OLs, we will be able to study how electrical activity governs myelination.<br/>We used silicon on insulator wafers with 4 mm thick device layer, and a silicon mesh was fabricated using photolithography and RIE. The mesh was comprised of 4 mm thick ribbons, mimicking axons, and 30 mm round nodes, mimicking somas. Then, hydrofluoric acid (HF) was used to remove the SiO2 under the device, exposing 70 mm long ribbons with a 4 mm square cross-section. The 4 min HF exposure was long enough to remove the oxide bellow the “axons”, while leaving some oxide bellow the larger “somas” so that the device remains in place. We then used a combination of anisotropic KOH etching and isotropic stain etching (HF and HNO3) to round the corners of the square to achieve an axon like morphology of long ribbon with round cross-sections and nanoporous rough surface. Moreover, the nanoporous surface modulated the band structure of the silicon-electrolyte interface to generate a highly responsive optoelectronic “axon”. Then, another HF etching was performed to remove the device from the handle layer and the device was transferred to a 3D collagen gel. Transferring it to the collagen gel served two purposes, the cells were allowed to grow in a 3D microenvironment, and silicon AA were the only photo-responsive material (no handle) so that the electrical activity comes from the AA alone, which mimics neurons in the native developing brain. Then, OLs were isolated from neonatal rat brains and cultured on the devices. We then applied optical stimulation of the device, and showed how optical stimulation enhanced the formation of myelin on the AA as compared to non-stimulated controls.<br/>Overall, the new bioinspired AA we propose here allows the investigation of the role of electrical activity in the myelination process. This is done by enabling local electrical modulation that is originated from the AA itself, and not from an external stimulating electrode. This also lays the ground to induce local modulation of specific “axons” or even different location of the “axons” and study the resulting myelination outcome.