Several prokaryotic and eukaryotic intra- and inter- cellular processes are initiated and controlled by a communication pathway from stimulus, to cell surface receptor, to cell nucleus, to mRNA, to cytokine signaling agents and higher tissue response. We recognize that both prokaryotic and eucaryotic biological processes can be influenced through cell membrane receptor mechanisms. With NIH support, we will develop model cardiovascular biomaterials that (a) use surface-tethered ligands to promote macrophage adhesion and instigate a healing cascade [1-2], while (b) releasing bioactive molecules specifically selected to negate bacterial receptor-mediated adhesion. We have developed poly(ether urethane) materials, modified with poly(ethylene glycol) tethers that (1) eliminate random protein adsorption and (2) allow for surface modifications by cell adhesion promoting peptides and monoclonal antibody fragments. While all treatments promoted macrophage adhesion, some also promoted macrophage activation. Results also indicate that both peptide- and Mab-decorated PEU surfaces significantly enhanced bacterial adhesion and biofilm formation versus base material. Consequently, for biomaterials to attract macrophage without promoting bacterial infection, a means to negate bacterial adhesion is needed. Thus, we will over a three (3) year period, develop model biomaterials that biologically prevent bacterial colonization. Objectives are: 1. We will isolate and characterize the cognate receptor(s) that the bacterium, Staphylococcus epidermidis (SE), employs to bind to fibronectin (FN)-coated surfaces - i.e., FN binding receptors (FN-BR) - and we will generate monoclonal antibodies (Mabs) to the entire receptor and its FN-binding domains. 2. A single chain variable fragment (scFV) antibody will be engineered from the variable heavy and light binding domains of the monoclonal antibody (MabFNBR) produced above. We will verify that the scFV antibody (FVFNBR) has the ability to bind to the SE FNBR receptor and block SE bacterial adhesion. 3. PEU materials will be fabricated containing one of the bacterial adhesion receptor blocking molecules (the MabFNBR or scFV antibody FVFNBR). Rates of bacterial anti-adhesion molecule release as a function of the amount of therapeutic agent loaded and biomaterial preparation will be determined. SE bacterial attachment studies will be carried out as a function of the specific biomaterial preparation in question and fluid phase cell concentration; under controlled hydrodynamic conditions. We will quantify macrophage adhesion, cytokine production, and macrophage activation; both with and without the presence of bacteria.
