Mapping protein dynamics and their origin at biomaterial surfaces in vivo

修复或再生肌肉骨骼系统损伤的疗法通常涉及植入 合成材料来稳定组织或促进再生。然而，合成材料会导致异物 反应（FBR），这可能导致不良后果。我们设计有效治疗策略的能力， 减缓FBR的障碍是对触发FBR的分子机制的不完全理解。 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， 导致不可逆吸附并产生损伤相关分子模式（DAMP）， 炎症我们最近的研究表明，这种观点是不够的，相反，蛋白质与 表面动态和DAMP可能会出现从多个不同的来源。为此，本提案旨在 为了检验蛋白质在植入生物材料上的吸附是动态的（翻转 连续地并且随时间变化），并且FBR由源自血清的DAMP和来自血清的DAMP维持。 由募集的骨髓细胞产生的DAMP的连续产生。制定了两个具体目标 来验证这个假设特定目标#1将确定随时间推移的表面吸附蛋白的身份 在FBR中使用生物正交标记。这一目标将纳入蛋氨酸（Met）类似物 叠氮基高丙氨酸在野生型FBR期间的不同时间普遍标记新合成的蛋白质 植入的老鼠将对标记的和未标记的新合成的蛋白质进行定量， 用LC-MS/MS鉴定，以确定表面吸附蛋白的瞬时性质。具体目标#2 将确定表面吸附蛋白质的来源及其身份在FBR使用细胞特异性 生物正交标记这一目标将使用最近创建的小鼠品系，该品系在甲硫氨酰- tRNA合成酶（MetRS*），其使得Met类似物叠氮正亮氨酸的细胞特异性装载（通过Cre驱动器）成为可能 转化成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吸附蛋白质。当与LC-MS/MS结合时， 还将测定来自每种来源的吸附蛋白质。每个目标将研究硅树脂作为模型 植入物，具有疏水（天然表面）或亲水（等离子体处理）表面化学性质， 研究疏水性对蛋白质表面吸附动力学的影响。此外，蛋白质的一个子集 将测试来自LC-MS/MS结果的它们在体外活化巨噬细胞并充当DAMP的能力。在 总之，这个探索性的项目将利用最近开发的体内蛋白质标记技术来回答 关于触发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，并告知生物材料的合理设计，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，通过这种干预，FBR可以被 以前所未有的控制力和精确度阻止了这一切

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.