Engineering of an optimized acellular peripheral nerve graft
The long-term goal of this work is to engineer a nerve graft for therapeutic applications in peripheral nerve repair. Currently, surgeons use an autologous nerve graft when attempting to repair a peripheral nerve injury with a defect longer than a few millimeters. This approach, however, has several significant limitations, including loss of function at the site from which the donor nerve is extracted. This dissertation describes the creation of an acellular nerve graft with a well-preserved extracellular matrix (ECM), a significant step toward the creation of a replacement for the autologous nerve graft. To create the acellular graft, the effects of various detergents on peripheral nerve structure and protein composition were examined. That knowledge was subsequently used to develop a chemical process for deriving an acellular graft with a well-preserved ECM from native nerve tissue. The success of this process was demonstrated through histological and Western analysis of the graft. Subsequently, the graft was implanted into the sciatic nerve of a rat, and immunological tolerance to the graft was examined. The cellular response to the graft was compared to a positive control that was immunologically tolerated and a negative control that was rejected. The number of macrophages and T-cells present in the graft after 28 days illustrated that the optimized acellular graft was immunologically tolerated. The regenerative capacity of the optimized acellular graft was also examined in vivo. Acellular grafts created with the most common thermal and chemical decellularization methods were also tested. In initial studies, axon density at the midpoint of the optimized graft was 96% higher than in the thermally decellularized model and 42% higher than in the chemically decellularized model. The results imply that a well-preserved ECM and the removal of cellular material are both important for optimizing regeneration through an acellular nerve graft. In addition to serving as a model for studying the role of the ECM in regeneration, cells, growth factors, or both, can be incorporated into the optimized acellular graft. Thus, the graft may act as a natural scaffold upon which a clinical alternative to the autologous graft can be built.