Freeze dried, acellular nerve scaffolds with longitudinal channels for Peripheral Nerve Regeneration

Abstract

Nerve injuries can occur due to automobile accidents, gunshot injuries, thermal or electric shock and lacerations caused by sharp instruments. At least 2 million people worldwide suffer from peripheral nerve injuries annually, with estimated costs of $7 billion incurred due to paralysis alone. The current "gold standard" is the autograft?sensory nerves (eg. Sural nerve) harvested from other parts of the patient. However, this poses disadvantages such as loss of sensation at donor site, insufficient length of graft and high cost in addition to a low success rate of 50%. While several clinical alternatives composed of natural and synthetic biomaterials have been developed, none of them match the regenerative levels of the autograft. Allografts are nerves harvested from other human donors and may provide a viable alternative. However, patients who receive allograftsneed to undergo intense immunosuppression treatments due to host rejection of grafts. For this reason, it is of interest to remove cellular materialfrom the allografts. These acellular nerve grafts perform better than other clinically available nerve grafts but not as well as autografts. Current research on acellular nerve grafts focuses on the incorporation of additional components such as growth factors and cells that provide chemical guidance to regenerating axons.However, the pore size of acellular nerve grafts is ~10?m which does not allow effective penetration of cells or axons into the graft. In this study, we developed a decellularization protocol that sufficiently removed immunogenic cellular components without damaging the extracellular matrix. We then induced the formation of longitudinal channels of 20-60?m (diameter) inthese acellular grafts using a novel freeze drying process. These highly porous scaffolds exhibited similar tensileproperties asnative nerve tissue. Furthermore, when compared to acellular scaffoldsthat were not freeze dried, enhanced cell penetration and neurite outgrowth was observed with PC12 cells after 14 days of culture. The results of this study emphasize the importance of pore size on cellular infiltration. We therefore conclude that our novel freeze dried acellular scaffolds with longitudinal channelsserve as a basis for future peripheral nerve regenerative strategies using acellular allografts.

Nerve injuries can occur due to automobile accidents, gunshot injuries, thermal or electric shock and lacerations caused by sharp instruments. At least 2 million people worldwide suffer from peripheral nerve injuries annually, with estimated costs of $7 billion incurred due to paralysis alone. The current "gold standard" is the autograft'sensory nerves (eg. Sural nerve) harvested from other parts of the patient. However, this poses disadvantages such as loss of sensation at donor site, insufficient length of graft and high cost in addition to a low success rate of 50%. While several clinical alternatives composed of natural and synthetic biomaterials have been developed, none of them match the regenerative levels of the autograft. Allografts are nerves harvested from other human donors and may provide a viable alternative. However, patients who receive allograftsneed to undergo intense immunosuppression treatments due to host rejection of grafts. For this reason, it is of interest to remove cellular materialfrom the allografts. These acellular nerve grafts perform better than other clinically available nerve grafts but not as well as autografts. Current research on acellular nerve grafts focuses on the incorporation of additional components such as growth factors and cells that provide chemical guidance to regenerating axons.However, the pore size of acellular nerve grafts is ~10?m which does not allow effective penetration of cells or axons into the graft. In this study, we developed a decellularization protocol that sufficiently removed immunogenic cellular components without damaging the extracellular matrix. We then induced the formation of longitudinal channels of 20-60?m (diameter) inthese acellular grafts using a novel freeze drying process. These highly porous scaffolds exhibited similar tensileproperties asnative nerve tissue. Furthermore, when compared to acellular scaffoldsthat were not freeze dried, enhanced cell penetration and neurite outgrowth was observed with PC12 cells after 14 days of culture. The results of this study emphasize the importance of pore size on cellular infiltration. We therefore conclude that our novel freeze dried acellular scaffolds with longitudinal channelsserve as a basis for future peripheral nerve regenerative strategies using acellular allografts.