Genipin-Cross-Linked Chitosan Nerve Conduits Containing TNF-alpha Inhibitors for Peripheral Nerve Repair

摘要

Tissue engineered nerve grafts (TENGs) are considered a promising alternative to autologous nerve grafting, which is considered the "gold standard" clinical strategy for peripheral nerve repair. Here, we immobilized tumor necrosis factor-alpha (TNF-alpha) inhibitors onto a nerve conduit, which was introduced into a chitosan (CS) matrix scaffold utilizing genipin (GP) as the crosslinking agent, to fabricate CS-GP-TNF-alpha inhibitor nerve conduits. The in vitro release kinetics of TNF-alpha inhibitors from the CS-GP-TNF-alpha inhibitor nerve conduits were investigated using high-performance liquid chromatography. The in vivo continuous release profile of the TNF-alpha inhibitors released from the CS-GP-TNF-alpha inhibitor nerve conduits was measured using an enzyme-linked immunosorbent assay over 14 days. We found that the amount of TNF-alpha inhibitors released decreased with time after the bridging of the sciatic nerve defects in rats. Moreover, 4 and 12 weeks after surgery, histological analyses and functional evaluations were carried out to assess the influence of the TENG on regeneration. Immunochemistry performed 4 weeks after grafting to assess early regeneration outcomes revealed that the TENG strikingly promoted axonal outgrowth. Twelve weeks after grafting, the TENG accelerated myelin sheath formation, as well as functional restoration. In general, the regenerative outcomes following TENG more closely paralleled findings observed with autologous grafting than the use of the CS matrix scaffold. Collectively, our data indicate that the CS-GP-TNF-alpha inhibitor nerve conduits comprised an elaborate system for sustained release of TNF-alpha inhibitors in vitro, while studies in vivo demonstrated that the TENG could accelerate regenerating axonal outgrowth and functional restoration. The introduction of CS-GP-TNF-alpha-inhibitor nerve conduits into a scaffold may contribute to an efficient and adaptive immune microenvironment that can be used to facilitate peripheral nerve repair.