In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide(®)). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 μm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable artificial nerve guide for nerve tissue engineering.
Keywords: biodegradable nerve conduit; controlled release of GDNF; gelatin microspheres; gelatin-methacrylamide hydrogel; sciatic nerve regeneration.