Periodic Reporting for period 1 - MINECHPS-SCI (Development of minimally invasive neuromodulatory electroconductive hydrogel in combination with physical stimuli for spinal cord regeneration)
Periodo di rendicontazione: 2023-02-01 al 2025-01-31
Sintesi del contesto e degli obiettivi generali del progetto
The MINECHPS-SCI project addresses the debilitating impact of spinal cord injury (SCI), a condition that currently has no cure. With no effective treatments for functional recovery after injury, SCI remains one of the unresolved healthcare challenges till date. SCI affected 6.4 million people in Europe with an incidence of 0.19 million during 1990-2016. SCI, which is primarily caused by vehicle accidents, falls, and violence, affects the most productive age group (less then ~40 years old) of people. SCI disrupts connection between the brain and muscle, resulting in varying degrees of paralysis below the level of injury, including loss of movement, sensation, bowel and bladder control, and sexual function, as well as depression in 11-37% of patients. Current SCI management options are expensive, with a SCI patient in the United States incurring a staggering $2.4-5 million lifetime cost depending on the nature of the injury and yet not fully restoring functions.
Existing regenerative and rehabilitation approaches are largely palliative and neuroprotective, targeting the spared neural tissue after injury with limited efficacy in regeneration. While the spinal cord has poor regenerative ability, the degenerative processes further complicate the SCI pathophysiology. Hence, no regenerative and rehabilitation approaches, individually, have resulted in complete and near complete functional recovery to date. To fully restore the injured spinal cord's functionality, a coordinated combinational approach addressing particular degenerative processes is necessary. Our goal is to develop a minimally invasive, combinational approach to spinal cord regeneration (SCR) that combines regenerative methods with rehabilitation techniques to enhance functional outcomes. It involves the development of an injectable electroconductive hydrogel (ECH) with immunomodulatory effects that can induce regeneration by counteracting the degenerative processes after injury. The project integrates these regenerative approaches with rehabilitation techniques using electrical stimulation (ES) to maximize the functional regenerative outcome. The ECH will be used as a physical support for axonal growth, which can also deliver ES and biologics to cells at the same time. ES through the ECH will be used to stimulate the neurons and glial cells to enhance regeneration and neuroplasticity.
Existing regenerative and rehabilitation approaches are largely palliative and neuroprotective, targeting the spared neural tissue after injury with limited efficacy in regeneration. While the spinal cord has poor regenerative ability, the degenerative processes further complicate the SCI pathophysiology. Hence, no regenerative and rehabilitation approaches, individually, have resulted in complete and near complete functional recovery to date. To fully restore the injured spinal cord's functionality, a coordinated combinational approach addressing particular degenerative processes is necessary. Our goal is to develop a minimally invasive, combinational approach to spinal cord regeneration (SCR) that combines regenerative methods with rehabilitation techniques to enhance functional outcomes. It involves the development of an injectable electroconductive hydrogel (ECH) with immunomodulatory effects that can induce regeneration by counteracting the degenerative processes after injury. The project integrates these regenerative approaches with rehabilitation techniques using electrical stimulation (ES) to maximize the functional regenerative outcome. The ECH will be used as a physical support for axonal growth, which can also deliver ES and biologics to cells at the same time. ES through the ECH will be used to stimulate the neurons and glial cells to enhance regeneration and neuroplasticity.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
The work performed in the MINECHPS-SCI project was structured around three primary objectives:
• Development and characterization of injectable electroconductive hydrogels (ECHs) as a minimally invasive platform for spinal cord regeneration (SCR)
• Design of a non-invasive wireless electrical stimulation (WES) platform
• In vitro validation of the combinational regenerative-rehabilitation approach for neuroprotection and immunomodulation
The key scientific achievements under each objective are detailed below:
Development of injectable electroconductive hydrogel (ECH) and its physicochemical and biological characterization
We successfully designed multifunctional silk-based electroconductive hydrogels (ECHs) that self-assemble without chemical cross-linkers, utilizing the hydrophobic amino acid sequences in silk fibroin. These minimally invasive hydrogels, derived from Bombyx mori (mulberry silk) and Antheraea assamensis (Indian non-mulberry silk), were further enhanced by incorporating PEDOT:PSS, a conductive polymer with mixed ionic-electronic conductivity tailored to the properties of biological tissues. The storage modulus (2-4 kPa) and conductivity (~0.5-1 S/m) closely resemble the mechanical (0.1-3 kPa) and electrical properties (1-10 S/m) of the human spinal cord, making them highly suitable for neural applications. They had also optimized charge-transfer resistance and charge injection capacity ensure efficient and safe low-potential electrical stimulation of human induced pluripotent stem cell (hiPSC)-derived neuronal systems. These ECHs supported hiPSC-derived neural and cardiac cell viability and demonstrated non-inflammatory responses in human blood monocyte-derived macrophages. Additionally, the rheological properties of these silk-based ECHs were explored for bioprinting applications. A key outcome from this work was published in Journal of Biomedical Materials Research Part A, demonstrating the feasibility of injectable conductive hydrogels for neuronal network formation.
Design of a non-invasive wireless electrical stimulation (WES) platform using silk based ECHs
We developed a WES platform with cell laden silk-based conductive hydrogel as the receiver and insulated copper sheet acting as the power transmitter and demonstrated its functionality to modulate macrophage behaviour and influence hiPSC-derived astrocytes and cardiomyocytes. This non-invasive yet highly effective WES setup relies capacitive coupling between the transmitter and receiver through electrostatic induction, facilitated by a dielectric medium composed of polystyrene and glass coverslip, that prevents direct electrical conduction but enables efficient energy transfer. A biphasic 2V pulse with 10 kHz, applied to the copper sheet, generated a corresponding alternating current ~100 µA and an electric field of ~300 mV in the conductive hydrogel. Due to its mixed ionic-electronic conductivity, our silk-based hydrogel formulation stands out as an efficient WES platform employing capacitive coupling mechanism, generating comparable outputs to other systems that require much higher energy inputs (MHz frequencies) to achieve the same.
In vitro validation of impact of the combinational regenerative-rehabilitation approach on macrophage polarization
In order to assess the potential immunomodulatory effect of the developed ECH in conjunction with the novel electrostatic driven capacitive coupling wireless electrical stimulation, we evaluated the polarization behaviour of human blood monocyte derived macrophages under the inflammatory environment. The initial findings demonstrated WES platform successfully delivered electrical stimulus to the macrophages seeded on the silk based ECH formulations without having any pro-inflammatory impact on the macrophages. Most importantly, under in vitro inflammatory environment (in presence of LPS), the WES significantly lowers the expression of inflammatory cytokines (IL-6, TNF-a), indicating the potential of the approach in regulating the neuroinflammation after SCI.
In vitro validation of impact of the combinational regenerative-rehabilitation approach on neuroprotection
Spinal cord injury (SCI) is a complex condition involving glial scar formation, vascular disruption, chronic inflammation, cell death, and growth factor deficiency. Ongoing research continues to unravel these intricate mechanisms. The dual and often contradictory roles of glial cells (astrocytes, microglia, oligodendrocytes) further complicate SCI pathology. Consequently, treatment strategies have evolved from single-target approaches to combinational therapies that address multiple pathological factors to enhance spinal cord regeneration (SCR).
Recent studies suggest that electrical stimulation (ES) plays a crucial role in modulating glial responses, highlighting its potential as a therapeutic intervention. Understanding the interactions between glial cells and neurons under ES is essential for creating a supportive microenvironment for neural repair. Astrocytes, the most abundant spinal cord cells, regulate CNS homeostasis, synaptic activity, and vascular flow while responding to physical stimuli like ES. Recent evidence indicates that ES can reduce astrocyte hyperreactivity post-SCI, lowering barriers to axonal regeneration and promoting tissue remodeling. Building on this, we investigated hiPSC-derived cortical astrocytes embedded in electroconductive hydrogels (ECHs). Our findings confirmed long-term cell viability and enhanced expression of connexin 43 (CX43) under the WES protocol, supporting the potential of ES-based regenerative strategies. We also induced reactive cortical astrocytes using microglial secretion factors, following an established protocol, and assessed how electrical stimulation (ES) modulates their hyperreactivity. Our findings suggest that ES may play a crucial role in neuroprotection following SCI by regulating astrocytic responses.
• Development and characterization of injectable electroconductive hydrogels (ECHs) as a minimally invasive platform for spinal cord regeneration (SCR)
• Design of a non-invasive wireless electrical stimulation (WES) platform
• In vitro validation of the combinational regenerative-rehabilitation approach for neuroprotection and immunomodulation
The key scientific achievements under each objective are detailed below:
Development of injectable electroconductive hydrogel (ECH) and its physicochemical and biological characterization
We successfully designed multifunctional silk-based electroconductive hydrogels (ECHs) that self-assemble without chemical cross-linkers, utilizing the hydrophobic amino acid sequences in silk fibroin. These minimally invasive hydrogels, derived from Bombyx mori (mulberry silk) and Antheraea assamensis (Indian non-mulberry silk), were further enhanced by incorporating PEDOT:PSS, a conductive polymer with mixed ionic-electronic conductivity tailored to the properties of biological tissues. The storage modulus (2-4 kPa) and conductivity (~0.5-1 S/m) closely resemble the mechanical (0.1-3 kPa) and electrical properties (1-10 S/m) of the human spinal cord, making them highly suitable for neural applications. They had also optimized charge-transfer resistance and charge injection capacity ensure efficient and safe low-potential electrical stimulation of human induced pluripotent stem cell (hiPSC)-derived neuronal systems. These ECHs supported hiPSC-derived neural and cardiac cell viability and demonstrated non-inflammatory responses in human blood monocyte-derived macrophages. Additionally, the rheological properties of these silk-based ECHs were explored for bioprinting applications. A key outcome from this work was published in Journal of Biomedical Materials Research Part A, demonstrating the feasibility of injectable conductive hydrogels for neuronal network formation.
Design of a non-invasive wireless electrical stimulation (WES) platform using silk based ECHs
We developed a WES platform with cell laden silk-based conductive hydrogel as the receiver and insulated copper sheet acting as the power transmitter and demonstrated its functionality to modulate macrophage behaviour and influence hiPSC-derived astrocytes and cardiomyocytes. This non-invasive yet highly effective WES setup relies capacitive coupling between the transmitter and receiver through electrostatic induction, facilitated by a dielectric medium composed of polystyrene and glass coverslip, that prevents direct electrical conduction but enables efficient energy transfer. A biphasic 2V pulse with 10 kHz, applied to the copper sheet, generated a corresponding alternating current ~100 µA and an electric field of ~300 mV in the conductive hydrogel. Due to its mixed ionic-electronic conductivity, our silk-based hydrogel formulation stands out as an efficient WES platform employing capacitive coupling mechanism, generating comparable outputs to other systems that require much higher energy inputs (MHz frequencies) to achieve the same.
In vitro validation of impact of the combinational regenerative-rehabilitation approach on macrophage polarization
In order to assess the potential immunomodulatory effect of the developed ECH in conjunction with the novel electrostatic driven capacitive coupling wireless electrical stimulation, we evaluated the polarization behaviour of human blood monocyte derived macrophages under the inflammatory environment. The initial findings demonstrated WES platform successfully delivered electrical stimulus to the macrophages seeded on the silk based ECH formulations without having any pro-inflammatory impact on the macrophages. Most importantly, under in vitro inflammatory environment (in presence of LPS), the WES significantly lowers the expression of inflammatory cytokines (IL-6, TNF-a), indicating the potential of the approach in regulating the neuroinflammation after SCI.
In vitro validation of impact of the combinational regenerative-rehabilitation approach on neuroprotection
Spinal cord injury (SCI) is a complex condition involving glial scar formation, vascular disruption, chronic inflammation, cell death, and growth factor deficiency. Ongoing research continues to unravel these intricate mechanisms. The dual and often contradictory roles of glial cells (astrocytes, microglia, oligodendrocytes) further complicate SCI pathology. Consequently, treatment strategies have evolved from single-target approaches to combinational therapies that address multiple pathological factors to enhance spinal cord regeneration (SCR).
Recent studies suggest that electrical stimulation (ES) plays a crucial role in modulating glial responses, highlighting its potential as a therapeutic intervention. Understanding the interactions between glial cells and neurons under ES is essential for creating a supportive microenvironment for neural repair. Astrocytes, the most abundant spinal cord cells, regulate CNS homeostasis, synaptic activity, and vascular flow while responding to physical stimuli like ES. Recent evidence indicates that ES can reduce astrocyte hyperreactivity post-SCI, lowering barriers to axonal regeneration and promoting tissue remodeling. Building on this, we investigated hiPSC-derived cortical astrocytes embedded in electroconductive hydrogels (ECHs). Our findings confirmed long-term cell viability and enhanced expression of connexin 43 (CX43) under the WES protocol, supporting the potential of ES-based regenerative strategies. We also induced reactive cortical astrocytes using microglial secretion factors, following an established protocol, and assessed how electrical stimulation (ES) modulates their hyperreactivity. Our findings suggest that ES may play a crucial role in neuroprotection following SCI by regulating astrocytic responses.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
The findings of MINECHPS-SCI project have advanced the field of spinal cord repair and bioelectronic medicine by demonstrating the proof of concept of novel silk based biocompatible and non-immunogenic ECHs and a WES platform, which can address the current limitations in treatment strategies following SCI. The project results beyond the state of art, potential impacts, future scope, etc. are outlined below .
Scientific and technological progress beyond the state of the art:
In contrast to conventional pre-formed scaffolds or passive biomaterials, we engineered self-assembling silk based ECHs without any chemical cross-linkers that can mimic the bioelectrical and dynamic spinal cord microenvironment. The unique mixed ionic-electronic conductivity enables the efficient bioelectric interfacing with neural tissue with high charge injection capacity and charge transfer efficiency. The ECH formulations developed in MINECHPS-SCI offer customizable flexibility, allowing their use as both an injectable hydrogel and a potential bioink. They can be engineered with tunable biodegradability, stiffness, and conductivity, making them a versatile and multifunctional conductive hydrogel platform for various biomedical applications. The WES platform developed in this project overcomes the limitations of existing ES approaches, which either require invasive electrode implants with risks of infection and degradation or rely on high-energy (MHz) wireless systems that are inefficient for long-term use. Instead, the WES system enables non-invasive, low-energy ES through electrostatic induction-driven capacitive coupling to silk-based ECHs, operating at 10 kHz biphasic pulses to effectively modulate neural and immune responses. Overall, we demonstrated a combined regenerative-rehabilitation approach for SCR by integrating biomaterials, ES, immunomodulation, and regeneration in a single platform. For the first time, we demonstrated that WES using injectable silk-based ECHs can regulate astrocyte hyperreactivity, a key barrier to SCR after injury, while also modulating inflammation by suppressing pro-inflammatory cytokines (IL-6, TNF-α) in human blood monocyte-derived macrophages. Thus, the project findings establish a multimodal approach for SCR providing a versatile and biologically responsive alternative to existing treatments.
Future Research Scope and Pathway to Commercialization
The project findings lay the foundation for developing next-generation minimally invasive bioelectronics for tissue engineering, designed to seamlessly integrate with the WES protocol for more efficient, safe, and stable electrical stimulation in biological environments. Additionally, it enhances our understanding of glial and macrophage responses to ES, opening new research avenues in neuroinflammation and immune-modulating biomaterials. With a strong potential for clinical translation, future research should focus on validating this approach in small and large animal models and optimizing ES parameters for different neural cell types through large-scale cell studies. In the long term, plans are in place to collaborate with medical device industries to accelerate commercialization, ensure intellectual property (IP) protection, and achieve compliance with medical regulatory standards (e.g. FDA approval). Additionally, a translational research proposal will be submitted through international collaboration with EU-wide consortiums, focusing on developing standardized bioelectronic interfaces for tissue engineering, including optimized ES protocols.
Scientific and technological progress beyond the state of the art:
In contrast to conventional pre-formed scaffolds or passive biomaterials, we engineered self-assembling silk based ECHs without any chemical cross-linkers that can mimic the bioelectrical and dynamic spinal cord microenvironment. The unique mixed ionic-electronic conductivity enables the efficient bioelectric interfacing with neural tissue with high charge injection capacity and charge transfer efficiency. The ECH formulations developed in MINECHPS-SCI offer customizable flexibility, allowing their use as both an injectable hydrogel and a potential bioink. They can be engineered with tunable biodegradability, stiffness, and conductivity, making them a versatile and multifunctional conductive hydrogel platform for various biomedical applications. The WES platform developed in this project overcomes the limitations of existing ES approaches, which either require invasive electrode implants with risks of infection and degradation or rely on high-energy (MHz) wireless systems that are inefficient for long-term use. Instead, the WES system enables non-invasive, low-energy ES through electrostatic induction-driven capacitive coupling to silk-based ECHs, operating at 10 kHz biphasic pulses to effectively modulate neural and immune responses. Overall, we demonstrated a combined regenerative-rehabilitation approach for SCR by integrating biomaterials, ES, immunomodulation, and regeneration in a single platform. For the first time, we demonstrated that WES using injectable silk-based ECHs can regulate astrocyte hyperreactivity, a key barrier to SCR after injury, while also modulating inflammation by suppressing pro-inflammatory cytokines (IL-6, TNF-α) in human blood monocyte-derived macrophages. Thus, the project findings establish a multimodal approach for SCR providing a versatile and biologically responsive alternative to existing treatments.
Future Research Scope and Pathway to Commercialization
The project findings lay the foundation for developing next-generation minimally invasive bioelectronics for tissue engineering, designed to seamlessly integrate with the WES protocol for more efficient, safe, and stable electrical stimulation in biological environments. Additionally, it enhances our understanding of glial and macrophage responses to ES, opening new research avenues in neuroinflammation and immune-modulating biomaterials. With a strong potential for clinical translation, future research should focus on validating this approach in small and large animal models and optimizing ES parameters for different neural cell types through large-scale cell studies. In the long term, plans are in place to collaborate with medical device industries to accelerate commercialization, ensure intellectual property (IP) protection, and achieve compliance with medical regulatory standards (e.g. FDA approval). Additionally, a translational research proposal will be submitted through international collaboration with EU-wide consortiums, focusing on developing standardized bioelectronic interfaces for tissue engineering, including optimized ES protocols.