Date of Award

Fall 2007

Document Type

Thesis

Degree Name

Master of Science in Biomedical Engineering - (M.S.)

Department

Biomedical Engineering

First Advisor

Bryan J. Pfister

Second Advisor

Michael Jaffe

Third Advisor

Treena Livingston Arinzeh

Abstract

Spinal Cord Injury (SCI) causes destruction and degeneration of axons in the white matter of the spinal cord, resulting in functional loss and paralysis. A successful treatment of SCI requires axons to regenerate across damaged regions. Current studies focus on identifying mechanisms to promote axon regeneration in lesions and have yet to be successful in preventing nerve degeneration due to scar tissue formation. Establishing axonal bridges over long distances of SCI lesions remains a challenge, resulting in poor functional recovery. Instead of relying on promoting axon regeneration into lesions, Pfister et al. has developed a transplantable nervous tissue construct spanned by stretch grown axon tracts. These tracts of living axons are intended to act as a bridge to facilitate axon outgrowth from the nerve construct to the host nerves over long SCI lesions. While axon stretch growth is fast and efficient, the current approach uses a two-dimensional (2D) culture system, posing a challenge for uniform distribution of DRG explants throughout the culture. This yields in a less than optimal number of axon tracts being stretched.

The research objective of this thesis is to increase the number/density of axons that are stretched grown by using three-dimensional (3D) cultures. This thesis work involves modification of the existing 2D axon stretch growth device to achieve axon growth in 3D cultures. The design includes separating two 3D hydrogel cell cultures using a porous nylon mesh to constrain each half of the culture. Optimal mechanical properties of the collagen hydrogel and nylon mesh pore sizes are tested for mechanical support, best axon outgrowth. and the number of axons available for stretch growth. Phase contrast and fluorescent microscopy are used to determine axon outgrowth in the hydrogel and through the nylon mesh. Live staining with fluorescent intracellular dyes and confocal microscopy are used to quantify the cross sectional areas of axon bundles stretched using the 2D device. This provides insight into developing a quantification method for axons grown in the 313 setup to determine the efficiency and the growth mechanism of axons in 3D cultures.

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