Date of Award

12-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Webb, Ken

Committee Member

Burg , Karen

Committee Member

Metters , Andrew

Committee Member

Wen , Xuejun

Abstract

Spinal cord injuries cost the United States $20 billion per year, with an existing patient population of 256,000 growing by an estimated 12,000 each year. Current clinical therapies for spinal cord injury are limited to spinal immobilization and realignment via traction, surgery, administration of methylprednisolone sodium succinate (MPSS) within eight hours post-injury, and rehabilitation exercises. While these therapies are important and may minimize damage and restore limited function, there is a dire clinical need for treatments to address the growing population of chronically-injured patients.
Varying degrees of axonal regeneration and functional recovery following spinal cord injury have been achieved in animal models by transplantation of glial cells from the peripheral nervous system and olfactory region. The recent identification of bioactive soluble and adhesive molecules produced by glial cells provides the opportunity to deliver these stimuli through biomaterial-mediated approaches, such as controlled release, gene therapy, and recombinant protein immobilization. The long-term objective of this project is a biomimetic, multi-factorial approach utilizing grooved fibers to restore structure and provide guidance for regenerating axons coupled with bioactive adhesive molecule delivery via immobilization to a hydrogel within the fiber grooves and controlled release of neurotrophic factors from the hydrogel. The implant design can serve as a platform for both in vitro and in vivo analysis of combination therapies for different injured nerve populations.
The first part of this research focused on cloning and expression of a bioactive 140kDa fragment of L1 neural cell adhesion molecule. L1 is a particularly attractive candidate for neural regeneration because it is critical for proper nervous system development and in vitro studies have demonstrated selectivity of neuron adhesion to L1 in the presence of astrocytes, which play a major role in nervous system inflammation. The second part of this research focused on the synthesis and purification of acrylated Tetronic macromers, and the development of Michael addition methods for hydrogel crosslinking and protein immobilization. In order to establish the feasibility of these hydrogels for neural regeneration, initial testing was conducted using NIH 3T3 fibroblasts and fibronectin because of the well-known RGD-dependent interaction. Results demonstrated that fibronectin encapsulation and surface-immobilization through acrylation of fibronectin positively influenced fibroblast spreading and proliferation. The last part of this research focused on evaluating neuronal cell line and primary neuron response to L1, evaluating cytocompatibility of T904-acrylate hydrogels with neural cells, and developing immobilization methods for L1. Results indicate that surface-immobilization of L1 to hydrogels may be the most promising method of bioactive cell adhesion molecule delivery for neural regeneration.

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