In Vitro Biological Response Tests

All the experiments were performed in accordance with the EU guidelines (2010/63/UE) and Italian law (Decree 26/14) and were approved by the local authority veterinary service and by our institution (SISSA) animal well-being committee (OBPA). Animal use was approved by the Italian Ministry of Health (no. 22DABNQYA), in agreement with the EU Recommendation 2007/526/CE. Organotypic cultures were obtained from the spinal cords of E12 embryonic mouse (C57BL) as previously reported.^32,33 We tested the biocompatibility of the PDMS-encapsulated LSMO when interfaced to the spinal explant growth by coculturing embryonic spinal cord and dorsal root ganglia (DRG) thin slices for 4 weeks in vitro. These organ slices represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties, and spinal cord resident cells are retained in a 3D fashion.³³ By the use of this model, we have recently shown that artificial scaffolds enriched with nanostructures sustain axonal regeneration and spinal segment rewiring.³² Such tests were predictive and instrumental for in vivo scaffold implantation.³⁴ Live calcium imaging was performed as previously reported;³⁵ briefly, organotypic spinal cords were loaded with cell permeable Ca²⁺ dye Fluo-4 AM (Molecular Probes); 11.6 μL of DMSO (Sigma-Aldrich) was added to the stock 50 μg of the dye; and cultures were incubated at 4 μM final concentration. After dye loading and de-esterification, cultures were maintained in extracellular saline solution of the following composition (mM): 150 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH; osmolarity 300 mOsm). Samples were mounted on an inverted microscope (Nikon Eclipse Ti–U). The dye was excited at 488 nm, and the emission was detected at 520 nm, and cultures were observed with a 40× objective (PlanFluor, 0.60 NA). Images were acquired every 150 ms using the ORCA-Flash4.0 V2 sCMOS camera (Hamamatsu) operating at binning 4. Drugs used: 25 μM bicuculline (GABAA receptor antagonist; Sigma-Aldrich, St Louis, Missouri, USA) and 2 μM strychnine (glycine receptor antagonist; Sigma-Aldrich) perfused for 20 min to weaken the synaptic inhibition and increase the synchronization. Finally, 1 μM TTX (a voltage-gated, fast Na⁺ channel blocker; Latoxan, Portes-lès-Valence, France) was added to confirm the neuronal nature of the recorded signals. Images were analyzed with ImageJ software (NIH), and the corresponding traces were extracted with Clampfit software (pClamp suite, 10.4 version, Axon Instruments, San Jose, CA, USA). Following calcium imaging, organotypic slices were fixed in 4% formaldehyde, prepared from fresh paraformaldehyde, in PBS 1× for at least 1 h. After fixation, slices were incubated for 10 min with 0.1 m glycine in PBS 1× and permeabilized for 1 h with 0.3% Triton-X-100 (Carlo Erba, Emmendingen, Germany) in PBS 1× added with 5% FBS (Gibco) and 4% BSA (Sigma-Aldrich) to prevent nonspecific binding of primary antibodies. Samples were subsequently incubated with primary antibodies overnight at 4 °C and with the secondary antibodies for 2 h at room temperature.³⁵ Mounting was performed with antifade medium Fluoromount (Sigma-Aldrich) on 1 mm thick microscope glass slides. The samples were incubated with the anti-β-tubulin III primary antibody (1:800, Sigma-Aldrich) and visualized with Alexa 594 antirabbit in goat as the secondary antibody (1:800, Invitrogen) and with antineurofilament H (SMI-32, 1:800, Biolegend) recognized by Alexa 488 antimouse in goat as the secondary antibody (1:800, Invitrogen). Nuclei were stained with DAPI (1:500, Invitrogen). A Nikon Eclipse Ti2 inverted microscope connected to an A1R confocal system (Nikon Instruments) was used to acquire stitched images (10× Plan Apo λ, 0.45 NA) to obtain a morphological insight of the stained spinal cord slice cultures.