The unique biochemistry of adECM allows neural stem cells to adhere and grow. It is important to note that high levels of rGO directly control cell fate by turning NE-4C cells and embryonic neural progenitor cells into neurons. Furthermore, primary astrocyte fate is also modulated, as increasing rGO boosts the expression of reactivity markers while unaltering the expression of scar-forming ones. The response to different neural cell lines, for example, PC12 and SH-SY5Y neural cells, exhibited adherence and survival on scaffold surfaces, indicating biocompatibility. Although differentiation was limited without additional factors like laminin, the metabolic activity remained within acceptable thresholds. Among the different formulations tested, a 3D foam with a composition of 50% adECM and 50% rGO (adECM-rGO) and a 3D foam consisting only of adECM were selected for in vivo implantation in a rat model based on the highly encouraging in vitro results.
To explore and optimise the electrostimulation parameters delivered to the scaffold during the in vitro cell cultures, a new device, “A multi-well graphene-multielectrode array device for in vitro 3D electrical stimulation and its fabrication method,” was developed by Graphenest and UAVR partners, who have signed an IP agreement, and an international patent application was published (WO 2023/209676 AI).
The start of in vivo studies was delayed due to unforeseen circumstances in obtaining ethical approvals to conduct these experiments. Nevertheless, once the approvals were secured, the predicted experiments were executed. In vivo studies validated the safety of the adECM and adECM-rGO scaffolds, with no adverse systemic reactions or toxicity observed post-implantation. Histological analyses revealed effective cell infiltration and integration within the host tissue, affirming the potential of these scaffolds for neural tissue engineering applications. The research further delved into the implantation of scaffolds in SCI models, noting the significant tissue integration and limited fibrous encapsulation, especially in adECM-rGO foams. Moreover, the macrophage-mediated uptake of rGO suggests a favourable biodegradation profile for these materials.
Following these results, the hemisection in rats at the 10th thoracic vertebrae SCI implantation without electrical stimulation (ES) was performed on 46 animals divided by different groups. The experiment combining the scaffold with the ES device was conducted on a limited number of animals; however, it allowed us to demonstrate the proof of concept. This demonstrates that animal withstand the electrostimulation device when implanted subcutaneously and linked to the scaffold located at the hemisection lesion at T10 through stainless steel wires. This innovative concept is being protected by the UAVR partner.
The NSS project has made substantial strides in SCI repair methodologies, with promising in vitro and in vivo outcomes. The scaffolds developed exhibit strong potential for aiding neural regeneration, backed by a robust biocompatibility profile. As the project transitions from foundational research to clinical applications, the insights gained from this work will inform the strategic direction and exploitation of the project's deliverables.
