Agarose hydrogel based three dimensional scaffolds for nerve regeneration

Autografts are widely used clinically to repair peripheral nerve gaps. However, this method is restricted by the limited availability of donor nerve grafts. Using a tissue-engineering approach, we designed three-dimensional (3D) scaffolds containing laminin-1 (LN-1) and nerve growth factor (NGF) for stimulating peripheral nerve regeneration in vitro and in vivo. LN-1 was covalently coupled to agarose using 1′ 1, carbonyldiimidazole (CDI). LN-1-modified agarose significantly enhanced neurite extension from 3D cultured embryonic day 9 chick dorsal root ganglia (DRGs) and PC 12 cells. 1,2-bis (10,12-tricosadiynoyl)-sn -glycero-3-phosphocholine based lipid microtubules were loaded with NGF, and embedded into agarose. The NGF gradients stimulated directional neurite extension from DRGs in agarose. A PC 12 cell based bioassay demonstrated that NGF-loaded lipid microcylinders could release physiologically relevant amounts of NGF for at least 7 days in vitro. The scaffolds were filled into polysulfone tubes to bridge a 10-mm long gap in the sciatic nerve of rats. Two months after implantation, the successful rate of regeneration, the total number of myelinated axons, the density of myelinated axons, and the relative gastrocnemius muscle weight were significantly higher in LN-1 and NGF containing scaffolds compared to controls. Regeneration in tissue-engineered scaffolds was comparable to that in autografts.; The 3D scaffolds have the potential to repair central nervous system (CNS) injuries. However, the regenerating nerves will have to contend with the glial scar at the distal interface in CNS. Glial scar plays a critical role in the regenerative failure in the CNS of adult mammals through the formation of mechanical or biochemical barriers. We report an in vitro model using layered agarose hydrogels to study behavior of growth cones from E9 chick DRGs at 3D interfaces that were mismatched in terms of their elasticity or chondroitin sulfate content. A mechanical barrier formed by the elasticity mismatch of layered agarose greatly influenced ability of neurites from DRGs to cross the 3D interface. Chondroitin sulfate B (CS-B) was covalently coupled to agarose through CDI chemistry. The CS-B-modified agarose at the interface significantly inhibited DRGs' neurites, and treating the gels with chondroitinase ABC or doping the gels with LN-1-coupled agarose overcame the inhibitory effects.