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A STEP FORWARD TO SPINAL CORD INJURY REPAIR USING INNOVATIVE STIMULATED NANOENGINEERED SCAFFOLDS

Periodic Reporting for period 3 - NeuroStimSpinal (A STEP FORWARD TO SPINAL CORD INJURY REPAIR USING INNOVATIVE STIMULATED NANOENGINEERED SCAFFOLDS)

Okres sprawozdawczy: 2022-04-01 do 2023-09-30

The mission of NeuroStimSpinal was to contribute to the development of a treatment for spinal cord injuries (SCIs). SCIs result in para- and tetraplegia due to the disruption of descending motor and ascending sensory neurons on a partial or complete basis.
The objective was to produce an innovative, stimulus-responsive scaffold capable of stimulating neural tissue repair following SCI.
The scaffold was composed of graphene-based materials (GBM) and decellularized human adipose tissue (adECM) and was intended to be implanted at the site of traumatic injury. By combining the scaffold with an electrical stimulation device, it was intended to promote neuron outgrowth and reconnection.
The project successfully met its objectives by developing a series of innovative scaffolds based on adECM and GBM, showing promising applications for neural tissue regeneration. The scaffolds encompassed 3D foams, nanofibrous scaffolds, hydrogels and bioinks. The NSS project advanced understanding of cellular interactions with 3D adECM/rGO foams.
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.
The market strategy for NSS assets has been defined according to the application potential of products and technologies developed.

Focused market segment - SCI repair:
At the end of the project, a core technology with a technology readiness level (TRL) of 4 has been developed, demonstrating that the technology meets the required objectives in the context of SCI repair. Clinical and preclinical testing could be further explored after completion of the project to allow the pursuit of TLR 6 in subsequent phases of development. To accomplish this, additional investment, either private or public (HE), will be necessary. The EIC program, through a transition project, may provide an adequate opportunity to achieve a TRL 6 considering the outcomes achieved so far, the significant unmet medical need, and the lack of appropriate treatment options (which could compete with the NSS concept). The NSS consortium, led by UAVR, will make efforts in this regard. (NSS scheme3).

Based on the technology developed under the scope of NSS, key partners are presently active in applying for national and trans-national research project proposals capable of supporting further development and maturation of the technology under development at NSS to target the therapeutic market related the SCI repair.

Extend market potential to other therapeutic intended uses:
The application of adECM based scaffolds could be potentially extended to other therapeutic related applications: NSS could enable regenerative medicine companies to implement such type of product into other therapeutic products outside the scope of SCI. This effort will be led by Tecnalia.

Other non-therapeutic applications, including in vitro related:
Additional exploitation potential could be created from NSS assets in non-therapeutic markets. Partners in NSS will assess the business potential and product viability on the following additional domains and markets:
•New market applications derived from the knowledge acquired in the synthesis and functionalization of GBM: NSS project could enable GNST to expand its business by including new products in their portfolio that could be used in printable electronics applications.
•NSS allowed the development of knowledge and methods concerning the formulation of novel bioinks and hydrogel-forming materials that could be potentially applied for in vitro applications, including screening in RUO markets, for example. In addition, new tools have been established that are applicable to in vitro dynamic cell culture and can be applied in multiple research and development contexts. This effort will be led by Tecnalia.

Overview of NSS assets:
1) An implantable spinal cord stimulation device and a biological bridge structure supporting and promoting axon growth to treat SCI
2) adECM-based bioinks
3) adECM-based injectable hydrogels
4) Methods for production and screen printing of Graphene-based conductive inks
5) A multi-well graphene-multielectrode array device for in vitro 3D electrical stimulation and its fabrication method
6) Innovative protocols to obtain 3D models of cytotoxicity and immune responses
Schematic representation of the methodologies steps projected to achieve SCI repair
Schematic representation of an implantable spinal cord stimulation device