| Summary: | Spinal cord injury, a traumatic disease characterised by a massive degeneration of neural tissue, was recently targeted for neuroregenerative interventions. Our approach is the development of pharmacologically pulsed neuroimplants using 3D collagen scaffolds hosting NSCs. We aim to monitor the properties of NSCs ex vivo and in vivo, using synthetic small molecules with neuroprotective and neurogenic properties. Synthetic, highly lipophilic CNS bioavailable small molecules, synthesized by our group (microneurotrophins), bind to neurotrophins receptors (Gravanis et al, Science Signaling, 2012, Calogeropoulou et al., J Med Chem., 2009). BNN27 can specifically interact with TrkA and p75NTR receptors activating specific signalling pathways controlling neuronal cell survival and neurogenesis (Charalampopoulos et al, PNAS, 2004, Lazaridis et al., PLoS Biol., 2011). We are seeding embryonic and adult mouse NSC on collagen 3D scaffolds of different composition (collagen, chondroitin-6-sulphate and gelatin) and construction (size of pores and stiffness), testing cell behaviour (survival, proliferation or differentiation) in basal conditions or pulsed with neurotrophins and/or microneurotrophins. Using the knock in sox2-egfp mice strain and fluorescence activated cell sorting (FACS) analysis, we obtain NSCs cultures with a sox2-positive population more than 90% pure. We evaluate specific markers of proliferation (ki67) and/or differentiation (GFAP for glial cells, Tuj1 for mature neurons and O4 for oligodendrocytes): we are currently testing the possible effect of BNN27 on proliferation of cortical NSCs in 2D cultures (increased numbers of ki67 positive cells up to 12%). The composition and the structure of 3D scaffolds seem to play a significant functional role: scaffolds with a combined composition such as 50% collagen/50% gelatin and 92% collagen/8% chondroitin-6-sulphate support NSC survival since they sustain sox2 expression and propagate neurosphere formation. On the other hand, sensory neurons (Dorsal Root Ganglia, DRGs) grew better in pure collagen scaffolds with less rigid structure. Our preliminary findings suggest that microneurotrophin BNN27 enhances the proliferation of NSCs in culture. Furthermore, the structure of collagen scaffolds heavily affects cellular properties, like stemness or differentiation.
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