The obiective of this basic project is to investigate a novel therapeutic approach involving protein crosslinking reactions for use with heterograft-derived biomaterials utilized in pediatric cardiac surgery. The overall hypothesis of this proposal is that triglycidyl amine (TGA) crosslinking and related reactions, including those with bisphosphonates, enhance biocompatibility, improve biomechanics, and inhibit heterograft heart valve deqeneration due to major modifications of the structural proteins of the extracellular matrix (ECM), and cellular interactions with the TGA crosslinked ECM with subsequent changes in gene expression. This is a translational project with the goal of creating innovative biomaterials for pediatric cardiac surgery. Collaborative interactions will occur with Project 2, the Clinical Core (C), Core D (Microarray), Core E (Morphology) and Core F (Biostatistics and Bioinformatics).
Aim 1. Mechanisms of TGA crosslinking: The focus here will be on TGA chemistry and biomaterial interactions with studies of reaction conditions, crosslink formation, and resultant biomechanical effects. We will also study the reactions, reaction kinetics, and net effects on crosslinking and biomechanics involving TGA and 2,2 mercaptoeothylamino-ethylidene-1, 1-bisphosphonate (MABP), a bisphosphonate custom synthesized by our group that can be covalently linked to TGA treated substrates to prevent both porcine bioprosthetic cusp and aortic wall calcification (see Progress Report).
Aim 2. To study the gene expression patterns, as well as cellular and ECM mechanisms responsible for TGA crosslinking conferring resistance to calcification: We will investigate TGA and TGA plus MABP inhibition of calcification as this relates to cellular interactions with the TGA-crosslinked ECM. We will focus on TGA-related up-regulation of osteopontin (OPN), an ECM protein associated with calcification inhibition, and down-regulation of tenascin-C (TNC), an ECM protein associated with heart valve calcification. We will use cell culture studies with sheep aortic valve interstitial cells (SAVIC) to model celI-ECM interactions comparing TGA with and without MABP to control substrates.
Aim 3. To investigate in vivo the mechanisms responsible for TGA-crosslinking plus MABP inhibiting heterograft calcification, enhancing biocompatibility, and improving biomechanical interactions. We will investigate in subdermal and circulatory models TGA-induced ECM structural protein modifications with resulting cellular ECM interactions that lead to changes in gene expression profiles with upregulation of OPN, with associated calcification inhibition, enhanced biocompatibility, and enhanced biomechanical properties. Clinical retrievals (calcified and noncalcified bioprostheses and homografts) will also be subjected to DNA-microarray analyses and immunohistochemistry studies to investigate correlates with the mechanisms of interest.
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