Engineering Fiber-Based Nervous Tissue Constructs for Axon Regeneration.

Biomaterial-based scaffolds used in nerve conduits including channels for confining regenerating axons and 3-dimensional (3D) gels as substrates for growth have made improvements in models of nerve repair. Many biomaterial strategies, however, continue to fall short of autologous nerve grafts, which remain the current gold standard in repairing severe nerve lesions (<20 mm). Intraluminal nerve conduit fibers have also shown considerable promise in directing regenerating axons in vitro and in vivo and have gained increasing interest for nerve repair. It is unknown, however, how growing axons respond to a fiber when encountered in a 3D environment. In this study, we considered a construct consisting of a compliant collagen hydrogel matrix and a fiber component to assess contact-guided axon growth. We investigated preferential axon outgrowth on synthetic and natural polymer fibers by utilizing small-diameter microfibers of poly-L-lactic acid and type I collagen representing 2 different fiber stiffnesses. We found that axons growing freely in a 3D hydrogel culture preferentially attach, turn and follow fibers with outgrowth rates and distances that far exceed outgrowth in a hydrogel alone. Wet-spun type I collagen from rat tail tendon performed the best, associated with highly aligned and accelerated outgrowth. This study also evaluated the response of dorsal root ganglion neurons from adult rats to provide data more relevant to axon regenerative potential in nerve repair. We found that ECM treatments on fibers enhanced the regeneration of adult axons indicating that both the physical and biochemical presentation of the fibers are essential for enhancing axon guidance and growth.

Controlled formation of cross-linked collagen fibers for neural tissue engineering applications.

Fibrous scaffolds engineered to direct the growth of tissues can be important in forming architecturally functional tissue such as aligning regenerating nerves with their target. Collagen is a commonly used substrate used for neuronal growth applications in the form of surface coatings and hydrogels. The wet spinning technique can create collagen fibers without the use of organic solvents and is typically accomplished by extruding a collagen dispersion into a coagulation bath. To create well-controlled and uniform collagen fibers, we developed an automatic wet spinning device with precise control over the spinning and fiber collection parameters. A fiber collection belt allowed the continuous formation of very soft and delicate fibers up to half a meter in length. Wet-spun collagen fibers were characterized by tensile and thermal behavior, diameter uniformity, the swelling response in phosphate buffered saline and their biocompatibility with dorsal root ganglion (DRG) neurons and Schwann cells. Fibers formed from 0.75% weight by volume (w/v) collagen dispersions formed the best fibers in terms of tensile behavior and fiber uniformity. Fibers post-treated with the cross-linkers glutaraldehyde and genipin exhibited increased mechanical stability and reduced swelling. Importantly, genipin-treated fibers were conducive to DRG neurons and Schwann cell survival and growth, which validated the use of this cross-linker for neural tissue engineering applications.