(265c) Proteomic Analysis of Immunogenic Proteins in Xenogeneic Heart Valve Bioscaffolds

A tissue engineered heart valve has the potential to fulfill many of the goals of an ideal replacement heart valve. The major steps in producing such a valve include formation of a scaffold followed by cellularization of the scaffold material. Two major types of scaffold material are being investigated: xenogenic bioscaffolds and synthetic polymer scaffolds. Xenogenic bioscaffolds have the advantage of being biomechanically sound and do not require tissue morphogenesis prior to implantation. However, xenogenic materials may exhibit antigenicity. Decellularization of xenogenic scaffolds has been used in an attempt to lessen the immune response to the material, but this approach can diminish the biomechanical attributes of the scaffold, reduce the extent of normal cellularization, and still may not eliminate the sources of antigenicity. We have argued that antigen removal, rather than decellularization, is the crucial step in producing an immunologically acceptable xenogenic scaffold. A critical first step in pursuing this goal is to determine the full extent and distribution of xenoantigens in these materials.

To accomplish this, a proteomic approach was used to identify immunogenic proteins in a candidate xenogeneic biomaterial, bovine pericardium (BP). Cytoplasmic and membrane proteins were extracted from BP using differential solubility and precipitation techniques. Extracted proteins were separated by either 1D or 2D gel electrophoresis. Western blots were performed using serum from rabbits collected before and after immunization with homogenized BP. Blots were probed with anti-rabbit IgG light chain specific secondary antibody. 1D blots were used to assess the extent and distribution of antibodies in the serum, while 2D immunoblots were used to identify specific protein antigens. Target proteins were assessed using MALDI-TOF/TOF mass spectroscopy. Protein identifications were made using Mascot searches against the NCBInr database. All 2D protein spots analyzed were found to be bovine origin. IgG-specific anti-BP antibodies were found in both pre- and post-immunization serum. In the pre-immunization serum, the distribution of these antibodies was mainly towards cytoplasmic proteins in BP. Rapid and persistent development of anti-BP antibodies was found post-immunization, directed mainly at membrane proteins. IgG antibodies are likely to be associated with a significant immune response directed towards the tissue, as IgG formation can only occur with concurrent Th-cell help. Thus the finding of IgG antibodies directed towards these antigens indicates a concurrent cell mediated response.