Principles of genetic engineering and recombinant DNA technology were applied to the modification and microbial synthesis of artificial extracellular matrix proteins, which were designed for eventual application in vascular prostheses. Proteins were synthesized, in which the elastin-like pentapeptide (VPGIG) was repeated alone or in a blocky structure with a cell binding domain derived from the CS5 region of fibronectin. To control physical and mechanical properties, unique amine functionality was added to the termini of these proteins by the incorporation of lysine-containing fusions. Detailed purification protocols were developed for the isolation of three CS5-containing proteins having the same primary sequence, yet distinct lengths, and one CS5-free protein. The success of these protocols was verified by SDS-PAGE, Western analysis, amino acid analysis, and, in two cases, NMR spectroscopy. The integrity of the elastin-like sequence was further verified by the presence of a lower critical solution temperature (LCST). Reacting the engineered proteins with glutaraldehyde resulted in crosslinked biomaterials that exhibited improved stability, with respect to solubility, and controllable mechanical response. The elastic moduli of these biomaterials was found to be inversely related to the protein masses from which they were derived, and approached modulus values of native elastin. Approximately, three out of every 4 amines participated in the crosslinking reaction, which was observed to be complicated one. Measurement of biological properties was limited to the evaluation of substrate specificity, with respect to cellular adhesion and spreading. Human umbilical vein endothelial cells (HUVECs) were observed to adhere to crosslinked, CS5-containing substrates at levels similar to that of fibronectin (FN) and at levels significantly greater than to the CS5-free control. HUVEC adhesion was found to be independent of blocking conditions. The binding mechanism did not involve the elastin binding protein, yet was one in which the CS5 domain participated. Human umbilical artery smooth muscle cells (UASMCs) behaved in the same manner and with an unknown binding mechanism. Glutaraldehyde reaction products were implicated as participants in a nonspecific binding mechanism. Due to the ambiguities introduced by glutaraldehyde, a bis-N-hydroxysuccinimidyl ester of polyethylene glycol was employed as a crosslinking agent. The aqueous reaction conditions, although controllable to a certain extent, through pH and temperature, proved to be deleterious in achieving stable networks. HUVEC seeding indicated that this strategy, or other similar ones, may not alter binding characteristics in a significant manner.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-3291 |
| Date | 01 January 1999 |
| Creators | Welsh, Eric R |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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