AbstractsBiology & Animal Science

Delayed wound healing in many chronic wounds has been linked to the lack of extracellular matrix (ECM) support and the degradation of fibronectin (FN) by an abnormally high protease level. The ECM is important in wound healing because it provides physical and chemical cues that direct tissue growth and development. FN is a key ECM protein that attracts and binds different molecules and cells and thereby supports biological responses associated with wound healing. The goal of my study is two fold: (1) To create an ECM analogue based on a composite of polyethylene glycol (PEG) hydrogels and FN binding domains and (2) To stabilize FN against proteolytic degradation by conjugating it to PEG. To address the first goal, I used Michael addition chemistry to covalently link the cell-binding domain of FN, III9−10, to PEG diacrylate and cross-linked the conjugate to PEG hydrogels. The conjugation of PEG to III9−10 was through cysteines in the affinity tag Glutathione S Transferase (GST). The conjugate of GST-III9−10 and PEG was characterized by: (i) Circular dichroism studies to determine secondary structure, (ii) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weight, and (iii) Ellmans reagent to determine the efficiency of PEG conjugation to GST-III9−10. The conjugate of PEG and GST-III9−10 had comparable secondary structure to GST-III9−10. SDS-PAGE studies showed that up to three PEG molecules were attached to one GST-III9−10 molecule. The efficiency of PEG conjugation was greater than 90% and occurred within 30 minutes after PEG diacrylate addition. Adhesion assays were used as a metric of biological activity. These assays demonstrated that on a molar basis cell adhesion and spreading were significantly higher on PEG hydrogels with GST-III9−10 than those with the commonly used arginine-glycine-aspartic acid (RGD) peptide. Scaffold rigidity is an important biological cues that influence xi cell responses. However, a comparative study of rigidity on GST-III9−10 and RGD functionalized PEG hydrogels demonstrated that differences in rigidity could not account for differences in cell adhesion and spreading between RGD and GST-III9−10 functionalized PEG hydrogels. Thus as ECM analogues, GST-III9−10 functionalized hydrogels provide more robust biochemical cues than RGD functionalized hydrogels. Studies of PEG hydrogel composites with mixtures of III9−10 and a fibronectin binding domain demonstrated that biological responses of adhesion and spreading and extracellular matrix assembly could be controlled by varying the ratio of these two domains and the rigidity of the PEG hydrogels. FN was stabilized against proteolytic degradation by covalent attaching it to PEG or by PEGylating it. FN was first isolated from human plasma by gelatin affinity chromatography and then PEGylated using two methods. The first method is to PEGylate human plasma fibronectin (HPFN) at cysteine residues with 3.4 kDa PEG diacrylate. The second method is to PEGylate HPFN at…