Abstract

Injuries and diseases of the central nervous system (CNS) are the most catastrophic and costly ailments occurring in humans. The neuronal damage at the injury site is most often irreversible. Biomaterial bridges bearing extracellular matrix (ECM) components have the potential of promoting and controlling nerve outgrowth in the damaged CNS. To assist in this endeavor, this multidisciplinary dissertation aims at studying neuron-ligand pathfinding at the molecular level by surface-sensitive analytical methods, optical microscopy, and with cell culture techniques.

In order to investigate the interaction of neuron growth and ligands, specific and careful preparation and characterization of the biomaterial substrates to which the neurons bind is an essential first step, one that is almost always lacking in the classical neuroscience field. Newly developed bioactive surfaces modified with ECM proteins and protein derived peptides were produced with good chemical and spatial control. Various surface analytical techniques such as contact angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were applied to characterize the surface chemistry and the spatial distribution of the modified substrates. Cell culture experiments were conducted to test and quantify neuron regeneration on different surfaces.

In order to reliably study neuronal growth activity in the absence of contaminating glial cells, dissociated and purified dorsal root ganglion (DRG) neuron preparations are a prerequisite. A new "slow centrifuge" method was found to be effective in eliminating non-neuronal cells.

The first direct AFM force measurements were conducted on living neurons. AFM Poisson statistical method was successfully applied to analyze the single-molecule bond-rupture force for the neuron integrin receptor-ligand systems. The fact that the individual FN-integrin receptor bond rupture force was smaller than the individual GRGDSY-integrin receptor force, while the same peptide resulted in 25% shorter neurite length than whole-protein FN under the same conditions, was consistent with the theory reported by others: neuron migration and outgrowth involves the formation of new attachments at the cell front and the breakage of attachment at the rear. Within our experimental conditions, the higher binding affinity observed between the GRGDSY-integrin receptor makes the process of breakage harder and/or slower for this system when compared to the FN-integrin receptor system.