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.

修复或再生对肌肉骨骼系统损害的疗法通常涉及植入 合成材料稳定组织或促进再生。但是，合成材料会诱导异物 响应（FBR），这可能导致不良结果。我们设计有效的治疗策略的能力 对触发分子机制的不完整理解，减轻FBR会受到阻碍 fbr。 FBR的当前教条假设血清蛋白吸附到生物材料表面并展开， 导致不可逆转的吸附并创建启动损伤相关的分子模式（潮湿） 炎。我们最近的研究表明，这种观点不足，而是蛋白质与 动态表面，潮湿可能来自多种不同的来源。为此，该提议的目的是 为了测试蛋白质在植入的生物材料上的吸附是动态的假设（翻转 持续和时间变化），并且FBR由从血清衍生的潮湿和 连续产生由募集的髓样细胞产生的潮湿。开发了两个具体目标 检验这一假设。特定的目标＃1将随着时间的推移确定表面吸附的蛋白的身份 在FBR中使用生物正交标记。这个目标将结合蛋氨酸（Met）类似物 在FBR期间，在野生型期间，在FBR期间的不同时间标记了偶氮霍明氨酸，以普遍贴上新合成的蛋白质 带有植入物的小鼠。标记和未标记的新合成蛋白将被量化并蛋白质 用LC-MS/MS鉴定，以确定表面吸附蛋白的瞬时性质。特定目标＃2 将使用细胞特异性确定表面吸附蛋白的起源及其在FBR中的身份 生物正交标记。该目的将使用最近创建的小鼠系，该系列在甲二烯基中具有点突变 tRNA合成酶（METRS*），可实现Met模拟偶氮蛋白酶的细胞特异性加载（通过CRE驱动器） 进入新合成的蛋白质。白蛋白-CRE和LYSM-CRE驱动程序将用于确定 分别从血清和髓样细胞中吸附的蛋白质。与LC-MS/MS结合时， 也将确定来自每个来源的吸附蛋白。每个目标都将调查硅酮作为模型 植入物具有疏水性（天然表面）或亲水性（血浆处理）的表面化学性质， 研究疏水性在表面吸附蛋白动力学方面的作用。另外，蛋白质的子集 从LC-MS/MS结果中，将测试其在体外激活巨噬细胞并充当潮湿的能力。在 摘要，这个探索性项目将利用最近开发的体内蛋白标签技术来回答 有关触发FBR的事件的基本问题。通过这种理解，这个项目将 产生新的假设，并告知生物材料的合理设计以控制表面吸附的潮湿。 长期，我们的目标是开发基于生物材料的治疗干预措施，FBR可以通过该干预措施。 以前所未有的控制和精度阻止。