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Protein matrices engineered for control of cell and tissue behavior
Genetic engineering of artificial extracellular matrix (ECM) proteins represents an exciting avenue for the preservation and control of the specificity of natural polymers on a molecular level. This work focuses on controlling the mechanical and cell adhesion properties of artificial extracellular matrices intended for vascular graft applications. Genetic engineering methods were used for the design and preparation of artificial proteins containing sequences chosen to mimic elements of the ECM. The cell-binding domains (CBDs) of fibronectin impart biologic function, while an elastin-like repeat, [(VPGIG)2(VPGKG)(VPGIG)2] provides mechanical properties and sites for covalent crosslinking. Three proteins were constructed using this design that differ only by the CBD. The CBDs chosen for the proteins were the CS5 domain, the scrambled CS5 (SC5) domain, and the CS1 domain and are denoted by the respective CBDs. The SC5 protein was prepared as a negative control for the CS5 protein to assure cell adhesion was mediated by the CS5 domain. The CS1 domain binds the same integrin as the CS5 with a 20–100 fold higher affinity. Bis(sulfosuccinimidyl)suberate and disuccinimidyl suberate were used to crosslink protein films for mechanical testing. By varying the amount of crosslinker and protein weight fraction, films were prepared with Young's moduli ranging from 0.06 MPa to 0.97 MPa. The molecular weight between crosslinks (Mc) was calculated to lie between 3,000 and 38,000. The moduli and Mc of the proteins span the range reported for natural elastin. Thiolating the lysines in CS5 with N-succinimidyl S-acetylthioacetate allowed investigation of the effect of disulfide crosslinking on the mechanical properties. The modulus of the disulfide crosslinked films was 0.48 MPa corresponding to a Mc of 6000. Adhesion studies of human umbilical vein endothelial cells (HUVEC) showed increased and stronger adhesion to the CS5 protein over the SC5 protein. HUVEC adhesion to the SC5 was similar to the negative control (BSA) with very low or no adhesion. HUVEC adhesion to the CS5 ranged from the same as fibronectin to lower than fibronectin as the detachment force was increased. These studies indicated adhesion was mediated by the CBD since the proteins were otherwise identical.
Biomedical research|Polymers|Chemical engineering|Cellular biology
Di Zio, Kathleen Ann, "Protein matrices engineered for control of cell and tissue behavior" (2003). Doctoral Dissertations Available from Proquest. AAI3078678.