Biocompatible Heterograft Biomaterials

这个基础项目的目的是研究一种新的涉及蛋白质的治疗方法 用于儿童心脏外科的异种移植物衍生生物材料的交联化反应。这一建议的总体假设是，甘油三胺(TGA)的交联和相关反应，包括那些与双膦酸盐的反应，由于细胞外基质(ECM)结构蛋白的重大修改，以及与TGA交联的ECM的细胞相互作用和随后基因表达的变化，增强了生物相容性，改善了生物力学，并抑制了异种移植心脏瓣膜的退化。这是一个翻译项目，目标是为儿童心脏手术创造创新的生物材料。将与项目2、临床核心(C)、核心D(微阵列)、核心E进行协作互动 (形态学)和核心F(生物统计学和生物信息学)。目的1.TGA交联剂的作用机制：本文将重点放在TGA化学和生物材料的相互作用上，研究反应条件、交联剂的形成以及由此产生的生物力学效应。我们还将研究反应、反应动力学和净效应对交联和生物力学的影响，包括 TGA和2，2-硫代乙硫氨基-1，1-双膦(MABP)，这是我们团队合成的一种定制双膦酸盐，可以共价连接到TGA处理的底物上，以防止猪生物假体尖端和主动脉壁钙化(见进展报告)。 目的2.研究TGA交联型钙化抗性的基因表达模式以及细胞和ECM机制：我们将研究TGA和TGA+MABP对钙化的抑制作用，因为这与细胞与TGA-交联型ECM的相互作用有关。我们将集中在TGA相关的上调骨桥蛋白(OPN)，一种与钙化抑制相关的ECM蛋白，以及下调TNC，一种与心脏瓣膜钙化相关的ECM蛋白。我们将使用绵羊主动脉瓣间质细胞(SAVEC)的细胞培养研究来模拟CELI-ECM的相互作用，比较TGA和不加MABP来控制底物。目的3.在体内研究TGA-交联剂+MABP抑制异种移植物钙化、增强生物相容性和改善生物力学相互作用的机制。我们将在皮下和循环模型中研究TGA诱导的ECM结构蛋白修饰以及由此产生的细胞ECM相互作用，这些相互作用导致OPN上调导致基因表达谱变化，从而抑制钙化、增强生物相容性和增强生物力学性能。临床取材(钙化和非钙化的生物假体和同种移植物)也将接受DNA微阵列分析和免疫组织化学研究，以调查与 利益机制。

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.