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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

Design, synthesis and characterization of artificial extracellular matrix proteins for tissue engineering

Panitch, Alyssa 01 January 1997 (has links)
Artificial extracellular matrix proteins were synthesized using recombinant DNA technology. These proteins were meant to mimic the internal elastic lamina of the tunica intima of muscular arteries for use in the design of small diameter ($<$6 mm) vascular prostheses. These proteins are composed of two blocks, an elastin-like block made up of repeats of the pentapeptide (VPGIG) and a cell binding segment which is composed of the CS5 region of fibronectin. Proteins of the following sequence were designed, synthesized and characterized: (CS5 (VPGIG)$\sb{40})\sb3$ and (CS5(VPGIG)$\sb{20})\sb5$. Successful synthesis and purification were confirmed by NMR spectroscopy and amino acid analysis. These artificial extracellular matrix proteins were designed with both controlled structural integrity and endothelial cell binding ability. The presence of a lower critical solution temperature (LCST) for both proteins suggests that the properties of the elastin like blocks were maintained. The endothelial cell binding ability of the CS5 region of fibronectin was also retained, as demonstrated through human umbilical vein endothelial cell (HUVEC) adhesion and spreading on the proteins, as well as through inhibition of this adhesion through preincubation of the HUVECs with soluble GREDVDY peptide which is known to inhibit $\alpha\sb4\beta\sb1$ integrin binding to CS5. Equivalent numbers of HUVECs adhered to (CS5(VPGIG)$\sb{20})\sb5$ and to fibronectin, although the number of cells well spread on the former was only approximately 55% of the number of those well spread on fibronectin.

Engineering the extracellular matrix: A novel approach to polymeric biomaterials

Welsh, Eric R 01 January 1999 (has links)
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.

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