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Chitosan (CS), a derivative of the naturally occurring biopolymer chitin, has been widely used in a variety of biomedical applications including peripheral nerve repair because of its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this work, bi-layer CS flat membranes were developed to be easily manipulated and rolled to obtain flexible nerve guidance channels (NGCs). These bi-layer membranes were prepared via solvent casting technique and their chemical composition was previously optimized to realize flexible membranes to be enwrapped for NGC formation. Two kinds of CS films cross-linked with different crosslinking agents were combined to produce scaffold structures with both good biocompatibility and the desired mechanical strength imparted by the inner and the outer layer, respectively. A tight connection between the two layers was achieved and the physicochemical and mechanical properties of CS bi-layer membranes were investigated. Additionally, bi-layer membranes were used for bridge implantation across 10-mm long median nerve defects in rats, and the outcome of peripheral nerve repair at 12 weeks post-implantation was evaluated by a combination of immunohistochemical and histological investigations.
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