Periodic Reporting for period 3 - RISEUP (Regeneration of Injured Spinal cord by Electro pUlsed bio-hybrid imPlant)
Periodo di rendicontazione: 2023-05-01 al 2025-04-30
To achieve the main goal of the RISEUP project, that is the development of an electro-pulsed bio-hybrid (EPB) device to support functional recovery after spinal cord injury (SCI), different specific objectives were pursued, covering both technological development and biological validation. On the technological side, the project included the design and fabrication of the EPB, the integration of a wireless system for power delivery and stimulation control, and the creation of numerical models to simulate electromagnetic fields and predict stimulation effects. On the biological side, the work focused on controlling the fate of induced neuronal stem cells (iNSCs) and mesenchymal stem cells (MSCs) through specific stimulation protocols combining microsecond pulses (μsPEFs) and direct current. The anti-inflammatory effects of the stimulation were also investigated. In addition, the study examined how electrical stimulation could influence neuronal activity and synaptic reorganization in mature neurons and spinal cord tissue cultures. The final step involved testing the EPB in a rat model of acute SCI to assess its effectiveness in promoting recovery.
During the final two years of the RISEUP project, several steps ahead have been taken, and the results obtained confirm the relevance and potential of μsPEFs stimulation in regenerative medicine and neuroinflammation. First, we demonstrated that ultrashort electrical pulses can effectively influence stem cell fate, promoting either proliferation or differentiation. This effect was consistently observed across different stimulation protocols characterized by increased intracellular calcium oscillations, thereby highlighting the role of calcium modulation as a driver of phenotypic outcomes.
Importantly, the project also provided the first evidence, both in vitro and in vivo, of the anti-inflammatory potential of microsecond pulses. In vitro studies showed that longer pulse durations reduced inflammatory marker expression in macrophages and enhanced the immunomodulatory properties of mesenchymal stem cells (MSCs). In vivo, stimulation led to an increased number of resting microglia among recruited immune cells, suggesting a dampened microglial reactivity. These results are particularly novel, given that the in vivo anti-inflammatory action of microsecond pulses had not been previously reported and appears to be well tolerated.
Technologically, a major achievement is the realization of the final EPB prototype, consisting in the fully flexible wireless device capable of delivering electrical pulses with tunable intensity and duration. The final EPB was tested in vivo.
All the experimental results have been accompanied by numerical modeling studies, that assessed fine tuned models that help to clarify the mechanisms underlying the stimulation outcome and supported the setting up of the stimulation intensities to be applied during the protocol.
Overall, the RISEUP project has successfully demonstrated the feasibility and therapeutic potential of using microsecond pulsed electric fields to modulate cellular processes and inflammatory responses. It has also delivered innovative technological tools with potential for clinical use and commercial development. Beyond its scientific and technological contributions, RISEUP has significantly supported the training and career development of young researchers, who played an active role in all phases of the project. These results establish a strong foundation for further research and future exploitation activities, marking the action as a successful step toward next-generation electroceutical therapies.
During the final two years of the RISEUP project, several steps ahead have been taken, and the results obtained confirm the relevance and potential of μsPEFs stimulation in regenerative medicine and neuroinflammation. First, we demonstrated that ultrashort electrical pulses can effectively influence stem cell fate, promoting either proliferation or differentiation. This effect was consistently observed across different stimulation protocols characterized by increased intracellular calcium oscillations, thereby highlighting the role of calcium modulation as a driver of phenotypic outcomes.
Importantly, the project also provided the first evidence, both in vitro and in vivo, of the anti-inflammatory potential of microsecond pulses. In vitro studies showed that longer pulse durations reduced inflammatory marker expression in macrophages and enhanced the immunomodulatory properties of mesenchymal stem cells (MSCs). In vivo, stimulation led to an increased number of resting microglia among recruited immune cells, suggesting a dampened microglial reactivity. These results are particularly novel, given that the in vivo anti-inflammatory action of microsecond pulses had not been previously reported and appears to be well tolerated.
Technologically, a major achievement is the realization of the final EPB prototype, consisting in the fully flexible wireless device capable of delivering electrical pulses with tunable intensity and duration. The final EPB was tested in vivo.
All the experimental results have been accompanied by numerical modeling studies, that assessed fine tuned models that help to clarify the mechanisms underlying the stimulation outcome and supported the setting up of the stimulation intensities to be applied during the protocol.
Overall, the RISEUP project has successfully demonstrated the feasibility and therapeutic potential of using microsecond pulsed electric fields to modulate cellular processes and inflammatory responses. It has also delivered innovative technological tools with potential for clinical use and commercial development. Beyond its scientific and technological contributions, RISEUP has significantly supported the training and career development of young researchers, who played an active role in all phases of the project. These results establish a strong foundation for further research and future exploitation activities, marking the action as a successful step toward next-generation electroceutical therapies.
The RISEUP project successfully integrated biological and engineering efforts across three main phases. In the first year (RP1), the team developed an initial design for the Electro Pulsed Bio-hybrid (EPB), featuring interdigitated gold electrodes on a PLA substrate, and studied calcium oscillations’ role in cell proliferation and differentiation. They also conducted preliminary biological and biocompatibility assessments. In the second year (RP2), focus shifted to improving the EPB’s flexibility and conductivity by adopting porous amorphous gold electrodes and exploring wireless energy harvesting; biological protocols for electrical stimulation were optimized to promote cell growth and axonal development. Detailed numerical models of cellular responses, including electroporation and calcium dynamics, guided stimulation parameter selection, complemented by in vitro immune response evaluations and in vivo biocompatibility tests. In the final phase (RP3), efforts centered on delivering a fully functional wireless EPB for implantation in rats, supported by dosimetric simulations confirming effective electric field induction. Preliminary in vivo experiments using wired devices demonstrated the device’s potential to modulate tissue and immune responses, with additional studies on neuron and immune cell reactions to stimulation protocols. Overall, the project progressed from prototype development to in vivo testing, advancing toward a wireless bioelectronic device for therapeutic applications.
The results are continuously disseminated not only to the scientific community through the participation of the researchers involved in the project to international conferences, but also to much wider audiences through press releases, interviews and presentations at initiatives such as the European Researchers' Night and the Pint of Science.
The results are continuously disseminated not only to the scientific community through the participation of the researchers involved in the project to international conferences, but also to much wider audiences through press releases, interviews and presentations at initiatives such as the European Researchers' Night and the Pint of Science.
The RISEUP project has achieved significant advancements in both scientific understanding and technological development. Key findings include demonstrating that microsecond electrical pulses can influence stem cell fate by inducing proliferation or differentiation, primarily through modulation of intracellular calcium oscillations. Additionally, these pulses exhibit anti-inflammatory effects both in vitro—where longer pulse durations reduce inflammation markers in macrophages and enhance MSC immunomodulation—and in vivo, by increasing resting microglia and decreasing microglia reactivity, with no adverse effects observed in animals. These novel insights suggest new therapeutic possibilities involving pulse-based interventions.
From an engineering perspective, the project developed advanced computational models for dosimetry, enhancing the understanding of electrical stimulus effects. It also resulted in a fully flexible, wireless device capable of delivering customizable electrical stimuli, which has been successfully tested in vivo at TRL4. This device’s unique porous design maintains conductivity under extreme bending (up to 180°), offering superior durability compared to existing electrodes and broad potential for applications beyond spinal cord injury, particularly in inflammation-related pathologies.
Overall, the project has initiated a new research avenue on ultrashort electrical pulses, while also delivering innovative, market-ready biomedical devices with promising therapeutic and commercial implications.
From an engineering perspective, the project developed advanced computational models for dosimetry, enhancing the understanding of electrical stimulus effects. It also resulted in a fully flexible, wireless device capable of delivering customizable electrical stimuli, which has been successfully tested in vivo at TRL4. This device’s unique porous design maintains conductivity under extreme bending (up to 180°), offering superior durability compared to existing electrodes and broad potential for applications beyond spinal cord injury, particularly in inflammation-related pathologies.
Overall, the project has initiated a new research avenue on ultrashort electrical pulses, while also delivering innovative, market-ready biomedical devices with promising therapeutic and commercial implications.