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治疗通常涉及植入 用来稳定组织或促进再生的合成材料。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 缓解FBR受阻于对触发FBR的分子机制的不完全了解 FBR。目前的FBR信条假设血清蛋白质吸附到生物材料表面并展开， 导致不可逆吸附并产生与损伤相关的分子图案(阻尼值)，从而引发 发炎。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，并且阻尼物可能来自多个不同来源。为此，这项建议旨在 为了检验蛋白质在植入的生物材料上的吸附是动态的假设(翻转 持续且随时间变化)，而FBR由来自血清和 由重新招募的髓系细胞产生的持续产生的湿润。制定了两个具体目标 来检验这一假设。特定目标#1将随着时间的推移确定表面吸附蛋白质的特性 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 叠氮高丙氨酸标记野生型FBR过程中不同时间新合成的蛋白质 植入器官的小鼠。标记的和未标记的新合成的蛋白质将被量化，并将蛋白质 用LC-MS/MS鉴定，以确定表面吸附蛋白质的瞬时性质。具体目标2 将确定表面吸附蛋白质的来源和它们在FBR中的特性 生物正交标记法。这个目标将使用最近创建的小鼠品系，该品系在甲硫基上有点突变- TRNA合成酶(MetRS*)，使甲硫氨酸类似物氮杂诺亮氨酸能够细胞特异性加载(通过Cre驱动程序) 变成新合成的蛋白质。白蛋白-Cre和LysM-Cre驱动程序将用于确定 分别从血清和髓系细胞中吸附蛋白质。当与LC-MS/MS联用时， 从每个来源吸附的蛋白质也将被确定。每个目标都将把硅胶作为模型进行研究 具有疏水性(天然表面)或亲水性(等离子体处理)的表面化学的植入物， 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 从LC-MS/MS结果将测试它们在体外激活巨噬细胞和作为阻尼剂的能力。在……里面 总结，这个探索性项目将利用最近发展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。通过这样的理解，这个项目将 产生新的假设，并为生物材料的合理设计提供信息，以控制表面吸附的阻尼。 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 以前所未有的控制和精确度阻止。

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.