Glass surfaces were patterned with cell-adhesive regions of laminin adhesive peptides YIGSR, RGD, and IKVAV, and cell-repulsive regions of poly(ethylene glycol) (PEG). The patterns were created by sputter-coating titanium and then gold onto glass coverslips through electron microscope grids. Gold surfaces were modified with cysteine-terminated peptides to have approximately 450 fmol/ cm2 of peptide incorporated on the glass coverslips as determined with radiolabeled CGYIGSR. Amine-functionalized glass coverslips were prepared using an amine-functionalized silane and then further modified with PEG-aldehyde by a Schiff base reduction. All surfaces were characterized by X-ray photoelectron spectroscopy and water contact angles. Hippocampal neurons, plated from a serum-free medium, adhered preferentially to peptide-functionalized surfaces over PEG-modified surfaces. Cell adhesion and neurite outgrowth were limited to the peptide region, demonstrating that neurite outgrowth could be directed by a combination of cell-adhesive and cell-repulsive cues.

Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury. Recently it has been shown that in the developing CNS, a combination of cell-adhesive and cell-repulsive cues guide growing axons to their targets. We hypothesized that by mimicking these guidance signals, we could guide nerve cell adhesion and neurite outgrowth in vitro. Our objective was to direct primary nerve cell adhesion and neurite outgrowth on poly(chlorotrifluoroethylene) (PCTFE) surfaces by incorporating alternating patterns of cell-adhesive (peptide) and nonadhesive (polyethylene glycol; PEG) regions. PCTFE was surface-modified with lithium PEG-alkoxide, demonstrating the first report of metal-halogen exchange with an alkoxide and PCTFE. Titanium and then gold were sputtered onto PEG-modified films, using a shadow-masking technique that creates alternating patterns on the micrometer scale. PCTFE-Au regions then were modified with one of two cysteine-terminated laminin-derived peptides, C-GYIGSR or C-SIKVAV. Hippocampal neuron cell-surface interactions on homogeneously modified surfaces showed that neuron adhesion was decreased significantly on PEG-modified surfaces and was increased significantly on peptide-modified surfaces. Cell adhesion was greatest on CGYIGSR surfaces while neurite length was greatest on CSIKVAV surfaces and PLL/laminin positive controls, indicating the promise of peptides for enhanced cellular interactions. On patterned surfaces, hippocampal neurons adhered and extended neurites preferentially on peptide regions. By incorporating PEG and peptide molecules on the surface, we were able to simultaneously mimic cell-repulsive and cell-adhesive cues, respectively, and maintain the biopatterning of primary CNS neurons for over 1 week in culture.

A thesis submitted in confonity mth the requirements for the degree of Masten of Applied Science. Graduate Department of Chemical Engineering and Applied Chemistry lnstitute of Biomaterials and Biomedical Engineering in the University of Toronto @Copyright by Samar Saneinejad (1 999) National Library 1+1 , , n a , Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliog raphic Services services bibiiographiques 395 Wellington Street 395. nie Wsllirrgton OttawaON K1AON4

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