Clinically, reconstruction of a nerve gap can be performed with a nerve graft (carrying the risk of donor site morbidity) or with nerve guidance conduits (NGC), which are only applicable for small nerve defects. Both options are associated with poor outcomes for patients. Failure of current NGC to support regeneration across large defects (> 3cm) are thought due to a lack of cellular/glial support to the regenerating nerve. We seek to optimise our NGC, POLYNERVE® (a microgrooved PLA/PCL hollow conduit) for larger nerve defects by adding an intraluminal scaffold seeded with adipose-derived stem cells (ASC). Self assembling peptides (SAP) are small peptide chains that assemble into controllable nanostructures and can produce synthetic hydrogels with biological yet predictable physical characteristics e.g. mimicking that of a nerve. SAP gels biodegrade to naturally occurring amino acids and are non-toxic. Although considered superior to hollow conduits, there are few commercially available conduits with an intraluminal filler and those that are contain bovine collagen which (due to its animal origin) suffer inconstant characteristics and risk disease transmission. We hypothesized that our SAP hydrogels when added to POLYNERVE® will support neural regeneration in vitro and in vivo superior to that of collagen filled conduits.
To assess regenerative capacity in vitro we cultured dorsal root ganglion tissue explants in positively charged SAP hydrogels of different stiffness (1-10Kpa) for 14 days with immunostaining for B3 tubulin, S100 and separately Phalloidin. Imaging was performed with confocal microscopy to assess neuronal sprouting and cellular migration into gels. For the in vivo portion of experimentation, composite conduits containing the optimal SAP hydrogel were fabricated and implanted into a 10mm sciatic nerve gap in Lewis rats for 6 weeks (n=3) with assessment of gross and histological regeneration at this timepoint. In preparation for future in vivo work incorporating SVF into the SAP filler we also assessed in vitro SVF viability in SAP hydrogels for 21 days with LIVE/DEAD staining.
In vitro: Three-dimensional neuronal sprouting and cellular migration into SAP hydrogels (G1=1-2.5Kpa) was evident in 3D at day 14 in vitro. Stiffer SAP hydrogels (G1=>2.5Kpa) did not show evidence of neuronal sprouting in 3D culture. LIVE/DEAD staining confirmed SVF viability in vitro, in 3D, up to day 21 and quantification of calcein fluorescence indicated cellular proliferation with a 7-fold increase in fluorescence over 14 days (p < 0.0001) in SAP with (G1=2.5Kpa). In vivo: At 6 weeks rats were culled (n=3) and the reconstructed segment of sciatic nerve injury excised with the conduit carefully removed. Complete nerve regeneration was evident in 33% of collagen conduits and 66% of conduits containing SAP hydrogel.
Softer SAP hydrogels supported 3D neuronal growth in vitro. In vivo, novel composite conduits containing the optimal SAP hydrogel enabled nerve regeneration over a 10mm sciatic nerve gap which was superior to bovine collagen-filled conduits. Softer SAP hydrogels support long-term SVF viability which will support our plans to repeat the above in vivo experimentation with SAP + SVF and over longer timepoints (12 weeks).