Bridging the gap: Angiogenesis and stem cell seeding of processed nerve allograft

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Project Summary High energy trauma is commonly associated with peripheral nerve injuries. These poly-trauma cases number in the tens of thousands annually.[4] While reconstruction of a 1-2 cm gap in a purely sensory nerve may be bridged with engineered nerve conduits, longer and larger diameter mixed nerves require autologous nerve grafts. Few expendable sensory nerves are available to use. They are often insufficient in quantity and harvest results in significant permanent donor site sensory loss. A readily available alternative with similar or better performance is needed. Currently, the most promising alternative is the processed nerve allograft. Providing an ideal scaffold for regenerating axons, it yields good outcome in short sensory nerve defects. However, in longer motor nerve defects functional outcomes are disappointing and further improvement of the allograft is necessary. In this study, we aim to improve results of such allografts by providing additional biological support in the form of undifferentiated or “Schwan cell-like” adipose-derived mesenchymal stem cells (AMSCs) and/or surgical angiogenesis provided by an enveloping fascial flap. We will correlate functional evaluation of muscle size, weight and strength with observed changes in gene expression, stem cell survival and migration, nerve electrophysiology, histomorphometry, and extent of immunologic response. We have previously described and validated the means to accurately quantify tetanic force in the rat and rabbit, and have shown its superiority to the sciatic function index (SFI).[5-7] We have demonstrated processed nerve allograft to provide better motor recovery than hollow nerve conduits in 1 cm rat sciatic nerve defects, and found filling of collagen nerve guides with a glycosaminoglycoside matrix to have little additional value.[8-10] Direct delivery of vascular endothelial growth factor (VEGF) was ineffective in improving motor recovery in this model.[11] We have also isolated adipose-derived mesenchymal stem cell (AMSCs) in rats and rabbits, and differentiated them to produce “Schwann cell-like” patterns of gene expression. We have developed an optimized processed nerve allograft (OPA) using elastase to minimize cellular debris and found cold storage to avoid adverse structural changes created by freezing. We have also labeled AMSCs with luciferase, using the resulting bioluminescence to image them in vivo. These preliminary studies provide the needed background for the proposed investigation. Our goals are four-fold: 1) to ask if differentiation of AMSCs seeded onto a decellularized nerve allograft differ with respect to survival, migration, “Schwann cell-like” gene expression and immune response to the graft, and to determine if any observed differences effect motor recovery; 2) to similarly evaluate the effect of surgical angiogenesis; 3) to test whether AMSCs combined with surgical angiogenesis are synergistic in all of these measures. 4) Finally, we will ask how AMSC and surgical angiogenesis perform relative to the autograft ‘gold standard’.