Mapping protein dynamics and their origin at biomaterial surfaces in vivo

绘制体内生物材料表面的蛋白质动力学及其起源

• 批准号: 10206869
• 负责人: Stephanie J Bryant
• 金额: $ 16.75万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10206869
• 关键词: Address; Adsorption; Albumins; Amino Acids; Biocompatible Materials; Cells; Chemistry; Clinical; Confocal Microscopy; Cre driver; Data; Development; Devices; Equipment Malfunction; Event; FDA approved; Foreign Bodies; Generations; Goals; Hydrophobicity; Implant; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Innate Immune System; Label; Lead; Link; Longevity; Macrophage Activation; Mass Spectrum Analysis; Membrane Proteins; Methionine; Methionine-tRNA Ligase; Methods; Modeling; Molecular; Mus; Musculoskeletal; Musculoskeletal System; Myeloid Cells; Natural regeneration; Nature; Necrosis; Patients; Pattern; Plasma; Point Mutation; Process; Production; Protein Dynamics; Proteins; Role; Serum; Serum Proteins; Signal Transduction; Silicones; Source; Surface; Techniques; Testing; Therapeutic Intervention; Time; Tissues; Wild Type Mouse; adverse outcome; analog; base; capsule; communication device; design; hydrophilicity; implantable device; implantation; in vitro Assay; in vivo; insight; macrophage; neutrophil; next generation; prevent; recruit; repaired; response; therapeutically effective

Therapies to repair or regenerate damage to the musculoskeletal system often involve the implantation of synthetic materials to stabilize tissues or promote regrowth. However, synthetic materials induce a foreign body response (FBR), which can lead to adverse outcomes. Our ability to design effective therapeutic strategies to mitigate the FBR is hampered by an incomplete understanding of the molecular mechanisms that trigger the FBR. The current dogma of the FBR assumes that serum proteins adsorb to biomaterial surfaces and unfold, leading to irreversible adsorption and creating damage-associated molecular patterns (DAMPs) that initiate inflammation. Our recent studies suggest that this view is insufficient and instead that proteins interact with surfaces dynamically and that DAMPs may arise from multiple different sources. To this end, this proposal aims to test the hypothesis that the adsorption of proteins onto implanted biomaterials is dynamic (turning over continually and changing in time), and that the FBR is maintained by DAMPs derived from serum and by the continuous generation of DAMPs that are produced by recruited myeloid cells. Two specific aims were developed to test this hypothesis. Specific Aim #1 will determine the identity of surface-adsorbed proteins over time in the FBR using bioorthogonal tagging. This aim will incorporate the methionine (Met) analog azidohomoalanine to ubiquitously tag newly synthesized proteins at different times during the FBR in wildtype mice with implants. The tagged and untagged newly synthesized proteins will be quantified and the proteins identified with LC-MS/MS to determine the transient nature of the surface-adsorbed proteins. Specific Aim #2 will determine the origin of surface-adsorbed proteins and their identity in the FBR using cell-specific bioorthogonal tagging. This aim will use a recently created mouse line that has a point mutation in methionyl- tRNA synthetase (MetRS*) that enables cell-specific loading (via Cre drivers) of the Met analog azidonorleucine into newly synthesized proteins. Albumin-Cre and LysM-Cre drivers will be used to determine the origin of the adsorbed proteins from serum and myeloid cells, respectively. When combined with LC-MS/MS, the identity of the adsorbed proteins from each source will also be determined. Each aim will investigate silicone as a model implant, having a surface chemistry that is either hydrophobic (native surface) or hydrophilic (plasma-treated), to study the role of hydrophobicity on the dynamics of surface-adsorbed proteins. In addition, a subset of proteins from the LC-MS/MS results will be tested for their ability to activate macrophages in vitro and act as DAMPs. In summary, this exploratory project will utilize recently developed in vivo protein labeling techniques to answer fundamental questions about the events that trigger the FBR. Through this understanding, this project will generate new hypotheses and inform the rational design of biomaterials to control surface-adsorbed DAMPs. Long-term, our goal is to develop a biomaterial-based therapeutic intervention through which the FBR can be prevented with unprecedented control and precision.

修复或再生肌肉骨骼系统损伤的疗法通常涉及植入 用于稳定组织或促进再生的合成材料。然而，合成材料会产生异物 反应（FBR），这可能导致不良后果。我们有能力设计有效的治疗策略 对触发 FBR 的分子机制的不完全理解阻碍了减轻 FBR FBR。目前 FBR 的教条假设血清蛋白吸附到生物材料表面并展开， 导致不可逆吸附并产生损伤相关分子模式 (DAMP)，从而启动 炎。我们最近的研究表明，这种观点是不够的，相反，蛋白质与 表面是动态的，并且 DAMP 可能来自多个不同的来源。为此，本提案旨在 检验蛋白质在植入生物材料上的吸附是动态的假设（翻转 持续且随时间变化），并且 FBR 由源自血清的 DAMP 和 由招募的骨髓细胞产生的 DAMP 持续生成。制定了两个具体目标 来检验这个假设。具体目标#1 将随着时间的推移确定表面吸附蛋白质的身份 在 FBR 中使用生物正交标记。该目标将纳入蛋氨酸 (Met) 类似物 叠氮高丙氨酸在野生型 FBR 期间的不同时间普遍标记新合成的蛋白质 带有植入物的小鼠。标记和未标记的新合成蛋白质将被定量，并且蛋白质 使用 LC-MS/MS 进行鉴定，以确定表面吸附蛋白的瞬时性质。具体目标#2 将使用细胞特异性确定表面吸附蛋白的来源及其在 FBR 中的身份 生物正交标记。该目标将使用最近创建的小鼠品系，该品系在甲硫氨酰-中具有点突变 tRNA 合成酶 (MetRS*)，可实现 Met 类似物叠氮正亮氨酸的细胞特异性加载（通过 Cre 驱动程序） 转化为新合成的蛋白质。白蛋白-Cre 和 LysM-Cre 驱动程序将用于确定 分别从血清和骨髓细胞中吸附蛋白质。与 LC-MS/MS 结合使用时，可鉴定 来自每个来源的吸附蛋白质也将被确定。每个目标都将研究硅胶作为模型 植入物，具有疏水性（天然表面）或亲水性（等离子体处理）的表面化学性质， 研究疏水性对表面吸附蛋白质动力学的作用。此外，蛋白质的一个子集 LC-MS/MS 结果将测试它们在体外激活巨噬细胞并充当 DAMP 的能力。在 总之，这个探索性项目将利用最近开发的体内蛋白质标记技术来回答 有关触发 FBR 的事件的基本问题。通过这种理解，该项目将 产生新的假设并为生物材料的合理设计提供信息以控制表面吸附的 DAMP。 从长远来看，我们的目标是开发一种基于生物材料的治疗干预措施，通过这种干预措施，FBR 可以 以前所未有的控制力和精确度进行预防。