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Neural Engineering

Neural Engineering - Poster Session D

Poster K2 - Optimizing Conduit Material Properties to Improve Nerve Regeneration

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Artificial nerve conduits are a growing focus in peripheral nerve repair, as they can direct repair following injury without the need for autologous nerve grafts. These conduits are difficult to study in vivo, so the development of novel methods of testing conduit materials in vitro is crucial for the advancement of the field [1]. This project involves the use of cervical spinal organoids to mimic nerve regeneration for the testing of different conduit materials, specifically focusing on the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) in a pHEMA hydrogel. In addition to its conductivity, PEDOT has been found to be both biocompatible and electrochemically stable, making it an ideal candidate for recording electrical signals in the body [2]. We investigate the hypothesis that axonal outgrowth will be both more substantial and more organized in conduits coated with PEDOT due to the enhanced mechanical strength provided by the PEDOT and the ability to mimic the typical electrical environment of nerves more closely.

Materials and

Methods:  To synthesize artificial conduits, the photocurable monomer poly(ethylene glycol)diacrylate (PEGDA) was molded into the desired structure using micro continuous liquid interface production (µCLIP) stereolithography. This process was used for the generation of both control PEGDA conduits and conduits integrated with PEDOT crystalline nanoparticles. The nanoparticles were distributed throughout the prints without any significant accumulations. The goal of this process was to create a conductive, biocompatible artificial nerve conduit that has the potential to interface between tissue and electronics.

Once synthesized, the conduits then had to be characterized to determine their conductivity and ability to adequately mimic the stiffness of a native nerve. The conductivity tests were performed using cyclic voltammetry on a PalmSens Impedance Analyzer. Samples were placed between two gold-plated electrodes fitted into a Swagelok. The compression tests were conducted on a DMA850 machine. After initial characterization, organoids were cultured in the conduits for approximately three weeks, using a PDL and laminin coat to promote adherence. The cells were then fixed and immunostained, and axonal growth was observed using fluorescence microscopy. The outgrowth of the axons was quantified using ImageJ software, where both the quantity and directionality were measured.

Results, Conclusions, and Discussions: Based on the characterization tests, the PEDOT conduits demonstrate desirable properties for nerve tissue integration. The conductivity of the PEDOT conduits is significantly higher than that of the control (Figure 1), which aligns with expectations as conductive nanoparticles provide a secondary pathway for charge carriers, enhancing ion flow. However, the compression tests placed the elastic modulus for PEDOT conduits at 700 Pa, below the desired range of 800-1600 Pa for intact nerves (Figure 2). This unexpected decrease in stiffness may be due to the nanoparticles disrupting the matrix of the base material. While the stiffness is not significantly outside the desired range, it does present an area for future development. The characterization methods proposed in this project offer a novel approach for quickly and efficiently testing conduit materials using an in vitro model, creating a helpful tool in the field of peripheral nerve regeneration research.

Regarding axonal growth, the PEDOT conduits demonstrated more dense and well-aligned outgrowth as compared with that of the control conduits (Figures 3, 4). The control conduits exhibit a sharp peak in the number of axons growing precisely at 0° along the conduits, likely due to structural ridges forcing alignment. The PEDOT conduits lack these ridges due to disruption from the nanoparticles. Accordingly, the peak for the PEDOT conduits is broader, suggesting that these conduits promote more natural axon growth without structural constraints. Additionally, control conduits show a secondary peak at 90°, indicating axonal wrapping around the conduit, which is detrimental to nerve regrowth. The PEDOT conduits do not show this trend, demonstrating improved directionality of growth. We conclude, therefore, that our methods of artificial nerve conduit synthesis produce conductive, strong pathways for neurons to grow along, demonstrating improvements in both length and directionality of axonal growth as compared to the control conduits. For the conduits to enhance nerve regeneration effectively, they must integrate seamlessly with the peripheral nervous system's complex electrophysiological signals. To test this, we plan to introduce a stimulation procedure to the conduits and observe the effects, expecting stimulated cells to exhibit more axonal growth in the conductive medium.

Acknowledgements (Optional):  [1] Mankavi F, Ibrahim R, Wang H. Advances in Biomimetic Nerve Guidance Conduits for Peripheral Nerve Regeneration. Nanomaterials. 2023; 13(18):2528. https://doi.org/10.3390/nano13182528

[2] Zhao, G., Zhou, H., Jin, G., Jin, B., Geng, S., Luo, Z., Ge, Z., & Xu, F. Rational design of electrically conductive biomaterials toward excitable tissues regeneration. Progress in Polymer Science. 2022; 131: 101573 https://www.sciencedirect.com/science/article/abs/pii/S0079670022000715