Abstract
Research suggests that regeneration of the spinal cord can occur if the microenvironment at the lesion site is pro-regenerative. Studies showed that injection of conditioned media from Mesenchymal stromal/stem cells (MSCs) cultures improved recovery after spinal cord injury as efficiently as injecting stem cells, which indicated that the main regenerative effects of MSCs result from paracrine mechanisms mediated by secreted factors, especially extracellular vesicles (EVs). A recent proteomic and RNA sequencing analysis of placental MSCs-derived EVs (PMSC-EVs) reveled several proteins and RNAs known to be involved in neuronal survival and development. However, it also has been reported that when systemically injected, EVs are cleared within 1-6 hours after administration, hence affecting their therapeutic effectiveness. Thus, in this study, to facilitate a long-term PMSC-EV neuroregenerative effect in spinal cord injury, we propose to design a novel delivery system that will allow for a local sustained release of PMSC-EVs to serve as a cell-free therapy to protect and regenerate neurons. We isolated PMSC-EVs and characterized them by using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western-blot. We immobilized the EVs onto two representative ECM scaffolds, injectable collagen hydrogel and small intestinal submucosa (SIS) patch material. Both are native ECM collagen-based scaffolds. We previously identified LLP2A, an integrin α4β1 ligand by one-bead one-compound (OBOC) combinatorial technology. We utilized a collagen-binding peptide, SILY, to modify the collagen-based scaffolds with the LLP2A ligand to immobilize PMSC-EVs to the scaffold surface via their α4β1 integrin. Binding of EVs on modified scaffolds were examined in vitro via scanning electron microscopy (SEM). Neurorescue and neurogenesis assays were further conducted to test the scaffolds function. The release of EVs was analyzed using NTA. SEM images of the scaffolds showed differences in EVs binding on scaffolds modified with or without SILY-(LLP2A)2 ligand. A control release analysis showed a stronger binding affinity and slower release of particles from scaffolds modified with SILY-(LLP2A)2. While further experiments are needed to better characterize the mechanisms of binding and to fine-tune the scaffold function, the results from this study suggest that our controlled release system has a promising potential for neurorescue and neuroregeneration and also can be widely used for other tissue regeneration applications.