Fig. 2

Dorsal root ganglia (DRG) axonal network growth is dependent on the osteoclasts extracellular vesicles (EV). A Characterization of osteoclast-derived EV by Western blot using CD81, CD63 and CD9 membrane markers [EV enriched fraction (EV) vs. EV-depleted supernatant (SN)]. Ponceau red staining showing the total amount of protein loaded. Transmission electron microscopy of osteoclast-derived EV (white arrows) by negative staining. Scale bar 500 nm. B Nanoparticle tracking analysis (NTA; NanoSight NS300) of the osteoclast-derived EV enriched fraction showing the concentration vs. size distribution (diluted in filtered PBS 1:500). Lines representing 3 runs. C Representative images of DRG treated with osteoclast secretome (OC) and EV-depleted osteoclast secretome (EV-dep). Staining for βIII tubulin, scale bar 500 µm. D Quantification of axonal sprouting area of DRG. Data represented as box and whiskers (median, whiskers represent minimum to maximum range), ****p ≤ 0.0001. E Representative images of DRG cultures in microfluidic devices. Nerve terminals exposed to complete osteoclasts secretome (OC) and EV-depleted osteoclasts secretome (EV-dep). Axons stained against βIII-tubulin; scale bar: 1 mm. F Quantification of the axonal growth using AxoFluidic algorithm. The data were given by the spatial dependence decay function \(f(x) = A \cdot \exp ( - x/\lambda )\) of the axons that can effectively cross the microchannels, where the constant A represents the entering in the axonal compartment, and λ the scale of spatial decay, as a measure to represent the length of the neurites. G Representative images of DRG cultures in the microfluidic platforms. Nerve terminals exposed to neurobasal control (NB) and osteoclast-derived EV (EV+). Axons stained against βIII-tubulin; scale bar: 1 mm. H Quantification of the axonal growth using AxoFluidic algorithm. The constant A represents the enter in the axonal compartment, and λ the scale of spatial decay, as a measure to represent the length of the neurites. Results are presented as bar ± SD, ns—non-significative; *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001. Each dot represents a microfluidic device analyzed from at least three independent experiments