(277g) Development of a Conduit System Enabling Controlled Release of Customized Exosomes and Wireless Electrical Stimulation for Peripheral Nerve Regeneration.

Peripheral nerve injuries (PNI) affect 2.8% of trauma patients worldwide, leading to long-term disability, reduced life quality, and heavy economic and social burdens (e.g., ~ $150 billion spent annually in the U.S. from over 200K procedures).¹ Despite several clinical treatment strategies with marginal success, incomplete recovery with poor functional outcomes are typical. The clinical gold standard, autologous nerve grafts, suffers from surgery at multiple sites, additional intraoperative electrical stimulation (ES), biological complexity, donor site morbidity, and limited length/size of graft tissue.^3-4 The implantation of native Schwann cells (SCs) via nerve guidance conduits (NGCs) has demonstrated promise. However, donor site morbidity, lack of availability, and slow in vitro growth of SCs limit clinical translation.^5-9 Alternatively, accessible and multipotent adult mesenchymal stem cells (MSCs) can be used in place of native SCs and autologously implanted via NGCs for PNI treatment.^10-12 However, recent studies showed that MSCs’ therapeutic effect depends on their ability to secrete bioactive molecules, in particular Exosomes (Exos), rather than the engraftment and differentiation at the injury site.^{13–18 14,19–23} The use of MSCs-derived Exos, in lieu of MSCs transplantation, has certain advantages; 1) higher safety profile, 2) lower immunogenicity, 3) ability to cross biological barriers, and 4) avoiding complications arising from cell differentiation and transplantation. However, challenges in obtaining Exos with necessary amount/therapeutic content and enabling local/sustained Exos delivery to the injury site limit clinically relevant treatment tools, specifically for large (>1 cm nerve gap) PNI. Therefore, the overall objective of this study is to 1) control MSCs transdifferentiation into SC-like phenotypes and modulate Exos bioproduction/content in a 3D graphene foam (3D-GF) using ES, 2) develop a biodegradable and implantable conduit system that can enable controlled release of e-Exos (with therapeutic content specifically customized for PNI) and 3) provide on demand wireless ES (WES) to promote guided axonal growth. Our hypothesis is that 1) ES can activate specific Exos bioproduction pathways (i.e., Ca²⁺ channel or PI3K/Akt dependent pathways) of MSCs and customize their therapeutic content (miRNAs/proteins associated with nerve regeneration), and 2) an Exos-laden conduit platform enables controlled Exos release and postoperative on-demand WES modulating endogenous cells, restoring downregulated neurotrophins, promoting guided axonal regeneration and functional recovery.

Gelatin enhanced 3D-GF (Gel-3D-GF) with improved mechanical/electrical properties was fabricated using chemical vapor deposition (CVD), temperature induced phase separation (TIPS) and chemical cross-linking. The modified foams were already characterized in terms of structure, material-cell interactions and biocompatibility. The gelatin modification did not change the electrical conductivity and 3D porous structure of the foams, however, provided significant improvement in mechanical properties enabling easy handling (facilitates suturing for in vivo surgeries). The MSCs were seeded within Gel-3D-GF and ES (100 mV/mm for 15 min per day for 10 days), free from any additional chemical induction (i.e., growth factors), was applied through conductive plates. ES resulted in significant enhancement in the expression of SC markers (i.e., S100, P75NTR, S100β, MPZ, Krox20 and MBP) at both protein and gene level demonstrated by immunocytochemistry (ICC), Western Blot (WB) and qPCR analysis while not affecting the cell viability and proliferation. The applied ES conditions as well as the Gel-3D-GF material did not alter the cell cycle or caused any apoptotic/necrotic cell population, reactive oxygen species (ROS) or DNA damage indicating in vitro biocompatibility. Our results also indicated that the MSCs to SCs transdifferentiation was modulated by the activation of Ca²⁺ channel signaling. In addition, we also collected the cell culture media (exosome depleted serum was used) during the ES directed transdifferentiation and isolated the Exos using commercially available exosome isolation kit. The isolated Exos (from control MSCs (c-Exos) and electrically stimulated MSCs (e-Exos)) was characterized in terms of amount (nanoparticle tracking analysis (NTA) and Exos quantification kit), size/shape (NTA and transmission electron microscopy (TEM)) and expression of exosome markers (ELISA and WB) as well as the exosomal protein and miRNA content (BCS protein and Qubit miRNA assay). Our results showed that both Exos (c- & e-Exos) have spherical shape with size range of 5-150 nm and expressed common exosomal markers (i.e., CD9, CD63, CD81, ALIX). However, the total Exos amount was significantly higher in electrically stimulated MSCs along with their protein and miRNA content (c-Exos amount: ~1x10⁹ , protein: ~20 µg/mL , miRNA: ~25 ng/µL & eExos amount ~1x10¹¹ , protein: ~40 µg/mL miRNA: ~50 ng/µL). This demonstrated the synergistic effect of ES and 3D structure on the Exos bioproduction. We also conducted a detailed proteomics and RNAseq analysis to understand how the ES modulates the transdifferentiation and associated Exos content. Our results indicated that the transdifferentiated MSCs have upregulated genes and proteins (i.e., MYL9, ACTN1, CAV-1, HSPB1, TBB4B, CTGF, TGFI1, ARF6, EZR, WNT5A etc.) that are associated with axonal regeneration and myelination upon ES which was aligned with the protein and miRNA content (i.e., miR340 and 221) of the e-Exos. This demonstrated that the obtained e-Exos has potential to be used as therapeutics to enhance the axonal regeneration upon PNI.

The e-Exos were then incorporated into gelatin portion of the Gel-3D-GF and crosslinked at different ratios using EDC-NIH chemistry to enable controlled release. The release profiles were constructed over time and 5-10 days of sustained release were observed. In the meantime, the graphene-based wireless flexible electronic cuff was already fabricated via our patented polymer casting/laser cutting method and characterized (US Patent 11,938,708 & US Patent 11,926,524). The e-Exos loaded Gel-3D-GF was integrated into the cuff structure via surgical glue and the entire system was characterized in terms of structure, integrity, stability, electrical properties and controlled release of Exos. The synergistic effect of Exos release and on-demand WES provided through the conduit system was evaluated on human motor neurons and native SCs via immunolabeling, molecular characterization, imaging and by identifying regulated genes/proteins associated with myelination, axonal guidance, and paracrine activity. Similar functional experiments were also performed on 3D ex vivo model using motor neurons and SCs. Our results indicated that in both vitro and ex vivo, the synergistic effect resulted in higher neurite extension in motor neurons and enhanced expression of neuronal and glial markers.

The surgical implantation procedure of the entire system on cadaver rats was performed to evaluate the feasibility of handling and suturing on sciatic nerve transection model. The system was successfully implanted. The pilot in vivo degradation and biocompatibility test was also performed and the results indicated that the implanted system maintained its integrity for 2 weeks and did not cause any major complications. The synergistic effect of Exos release and postoperative WES on axonal regeneration is currently being evaluated via detailed histochemical and functional recovery tests on a rat sciatic nerve transection model.

In conclusion, this study provided fundamental knowledge about the mechanistic of Exos bioproduction and therapeutic content using ES along with the synergistic effect of Exos release and on-demand WES on directing peripheral nerve regeneration. This system also offered an alternative postoperative approach via Exos-laden, biocompatible and implantable wireless conduit platform to treat PNI using patient’s own cells. Broadly, this knowledge potentially opens the door using MSCs-derived Exos for PNI treatment.