A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a...

Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical and topographical characteristics. Topographically, the combination of the treatments results in a fusion of the outer single silk fibers to a closed layer with a thickness ranging from about 40 to 75 μm. In contrast to the outer wall, the inner lumen (not treated with processing solvents) still represents the braided structure of single fibers. Mechanical stability, elasticity, and kink characteristics were evaluated with a custom-made test system. The modification procedure described here drastically improved the elastic properties of our tubular raw scaffold, favoring its use as a NGC. A cell migration assay with NIH/3T3-fibroblasts revealed the impermeability of the SF-NGC wall for possible invading and scar-forming cells. Moreover, the potential of the SF-NGC to serve as a substratum for Schwann cells has been demonstrated by cytotoxicity tests and live-dead stainings of Schwann cells grown on the inner surface of the SF-NGC. In vivo, the SF-NGC was tested in a rat sciatic nerve injury model. In short-term in vivo studies, it was proved that SF-NGCs are not triggering host inflammatory reactions. After 12 weeks, we could demonstrate morphological and functional reinnervation of the distal targets. Filled with collagen, a higher number of axons could be found in the distal to the graft (1678±303), compared with the empty SF-NGC (1274±146). The novel SF-NGC presented here shows promising results for the treatment of peripheral nerve injuries. The modification of braided structures to adapt their mechanical and topographical characteristics may support the translation of SF-based scaffolds into the clinical setting. However, further improvements and the use of extracellular matrix molecules and Schwann cells are suggested to enable silk tube based conduits to bridge long-distance nerve gaps.