Title:
Decellularized grafts with axially aligned channels for peripheral nerve regeneration.

Abstract:
At least 2 million people worldwide suffer annually from peripheral nerve injuries (PNI), with estimated costs of $7 billion incurred due to paralysis alone. The current "gold" standard for treatment of PNI is the autograft, which poses disadvantages such as high fiscal cost, possible loss of sensation at donor site and the requirement of two surgeries. Allografts are viable alternatives; however, intensive immunosuppressive treatments are often necessary to prevent host rejection. For this reason, significant efforts have been made to remove cellular material from allografts. These decellularized nerve grafts perform better than other clinically available grafts but not as well as autografts; therefore, current research on these grafts includes the incorporation of additional components such as growth factors and cells to provide chemical guidance to regenerating axons. However, effective cellular and axonal penetration is not achieved due to the small pore size (5-10μm) of the decellularized grafts. The overall objective of this study was to induce axially aligned channels in decellularized nerve grafts to facilitate enhanced cell penetration. The specific aims of this study were to optimize a decellularization method to enhance cellular removal, to induce axially aligned pore formation in decellularized grafts through a novel unidirectional freeze drying method, to study the bulk mechanical properties of these modified decellularized grafts and to assess cell penetration into these grafts. To this end we modified an existing decellularization protocol to improve cellular removal while preserving matrix structure in rat sciatic nerve sections. Standard freeze drying and unidirectional freeze drying were employed to impart the necessary pore architecture, and our results suggest that unidirectional freezing is a pertinent modification to the freeze drying process to obtain axially aligned channels. These highly porous scaffolds obtained using unidirectional freeze-drying possessed similar tensile properties to native nerve tissue and exhibited enhanced cellular penetration after 14 days of culture when compared to non-freeze dried and standard freeze-dried scaffolds. The results of this study not only highlight the importance of aligned pores of diameters ~20-60μm on cellular infiltration, but also presents unidirectional freeze drying as a viable technique for producing this required architecture in decellularized nerves. To the best of our knowledge, this study represents the first attempt to manipulate the physical structure of decellularized nerves to enhance cell penetration which may serve as a basis for future peripheral nerve regenerative strategies using decellularized allografts.