Neurotrophic factor combinations and extracellular matrix-based hydrogels for nerve regeneration

Deister, Curt Andrew
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The goal of this research was to help define a microenvironment that optimizes nerve regeneration. The research focuses on the effects of extracellular matrix composition and structure and on neurotrophic factor combinations. This dissertation describes the optimization of a combination of neurotrophic factors and of the composition of extracellular matrix-based hydrogels. These optimums are goals for the microenvironment of new nerve grafts. Collaborative work to decellularize human nerve tissue and create bioactive scaffolds is also covered. The decellularized tissue has a well preserved extracellular matrix structure which provides mechanical and chemical cues to regenerating axons. The combination of neurotrophic factors was a mixture of the neurotrophin nerve growth factor (NGF), the glial cell-line derived neurotrophic factor (GDNF) family ligand GDNF, and the neuropoietic cytokine ciliary neurotrophic factor v (CNTF). In an explant model, the combination of 50 ng/ml NGF, 10 ng/ml GDNF, and 10 ng/ml CNTF induced the highest level of neurite outgrowth at a 752 ± 291% increase over controls and increased the longest neurite length to 2031 ± 436 microns from 916 ± 203 microns for controls. The optimum concentrations of the three neurotrophic factors applied in combination corresponded to the optimum concentration of each factor when applied individually. Neurite extension from the explant model through extracellular matrix-based hydrogels made from mixtures of laminin, fibronectin, collagen 1, and hyaluronic acid was also studied. A co-gel made with 1.5 mg/ml collagen 1 and 1.5 mg/ml laminin was optimum in this study, resulting in outgrowth of 85.9 ± 9.3 % and length of 1532 ± 91 µm, versus 0 ± 10.0 % and 976 ± 32 µm for the controls. The hydrogel results suggest that interactions between hydrogel components are not significant. The decellularized human nerve exceeded the minimum levels of extracellular matrix preservation and removal of cellular proteins. The overall results suggest the use of a composite nerve graft using segments of decellularized nerve to provide a highly structured, laminin rich scaffold and delivering exogenous neurotrophic factors from an external reservoir as a next generation nerve graft.