Project Portugal 2030

Summary

In the vast majority of the cases, traumatic SCI, in companion dogs, typically results in severe neurological deficits and disabilities, causing loss of sensory and motor capabilities, as well as gastrointestinal and urinary tract, cardiac and respiratory distress. Our hypothesis posits that any effective therapeutic strategy aimed at restoring functionality after SCI should exploit axonal growth and neural plasticity, which need to be triggered by modulating 3 key events to overcome the aggressive environment in the injury site: 1) Resolve the chronic inflammatory processes; 2) Clear inhibitory molecules of axonal growth, namely CSPGs; 3) Activate axonal growth and plasticity whitin 3D environments along paths supported by increased neurotrophic support. Due to the complexity of these challenges a multidisciplinary and combinatorial approach is necessary to explore functional regeneration of SCI. Therefore, in the ReSPINE we propose the integration of stem cells secretome, hydrogels and electroactive materials into a single device - ESH - which we believe holds great potential to advance therapeutic strategies for SCI. This concept is built upon our previous work (Fig.1-5), in which we have shown that: 1) MSCs secretome reduces glial scar, degrade CSPGs, mitigates neuroinflammation, and provides neurotrohic support that stimulates axonal growth (PMID:29352743;35065953;38343771;27505621;32984278). 2) GAG-star-PEG hydrogels facilitates onsite or intrathecal delivery of MSCs secretome to SCI sites (PMID: 36827964); 3) PHBV microspheres, upon the adequate stimuli, can induce axonal plasticity in 2D and 3D culture systems of neuronal and glial cells (PMID:38364446). Having this in mind the ReSPINE program is structure around 4 main objectives: 1) Development and Characterization of ESH: Our focus is on creating ESH by integrating PHBV microspheres and ASCs secretome into a GAG-StarPEG hydrogel matrix. Characterization will evaluate physical, chemical, and mechanical properties, along with sustained release of the secretome. 2) Modulation of Axonal Growth, Neural Plasticity, and Glial Reactivity Driven by ESH: We will investigate ESH's impact on neural regeneration, particularly axonal growth and plasticity, using human spinal cord motor neurons derived from induced pluripotent stem cells (iPSCs) from the biobank we have established and extensively characterized in the lab. In vitro experiments will assess axonal elongation, guide growth cones, and modulate glial reactivity and neuroinflammation. 3) Study of Cellular and Regenerative Effects of ESH in a 3D SCI Model – in vitro: Using an advanced 3D SCI model, we will study ESH's cellular and regenerative effects in a physiologically relevant environment. Assessments will include changes in cellular morphology, viability, proliferation, differentiation, and biomarker expression associated with regeneration and inflammation. 4) Demonstration of In Vivo Proof of Concept: We aim to validate ESH's efficacy in vivo using animal models of spinal cord injury. Evaluations will include functional outcomes, histological changes, and molecular alterations post-ESH administration. We will assess improvements in locomotor function, pain alleviation, tissue repair promotion in thoracic and cervical injury models. Additionally, we will investigate its effects on respiratory function. In order to achieve these objectives ReSPINE is divided in 4 experimental tasks with looped feedbacks between them (Fig.8).