The role of macrophage subpopulations in the rejuvenation of fracture repair

随着年龄的增长，骨修复的速度变慢，增加了延迟愈合或不愈合的可能性。 这些并发症是通过外科手术治疗的，会导致严重的发病率甚至死亡， 尤其是在老年人中。在这里，我们将建立在我们以前的工作基础上，使用异慢性共生(其中两个 不同年龄的小鼠共享血液供应)，表明暴露在年轻的循环和年轻的 巨噬细胞使老年小鼠的骨折修复恢复活力。在我们的初步数据中，我们使用了细胞谱系追踪 骨折修复中巨噬细胞发育来源的分析和异生实验， 并发现这些细胞来自卵黄囊来源的细胞亚群。有趣的是，这些细胞驻留在 在骨骼修复过程中，它们通过血液循环被招募。随着老鼠年龄的增长，这一亚群细胞 就会耗尽。在这项建议中，我们研究了这种细胞群体的作用及其在 通过实现以下目标使骨折修复恢复活力： 1)确定卵黄囊祖细胞来源的巨噬细胞在卵黄囊再生中的作用 骨折修复。将调查这些细胞可被标记或耗尽的异慢性异种共生现象 为了确定这群巨噬细胞中的年轻细胞对提高质量的贡献 在年长的动物身上修复骨折。 2)确定在独特的巨噬细胞亚群中表达的基因的功能 骨修复中的幼鼠：我们使用单细胞RNA测序，发现了一个独特的亚群 巨噬细胞只存在于幼年动物的骨修复中。缺乏编码基因的小鼠 不同巨噬细胞群体中的分泌蛋白将用于异慢性异种共生以确定 他们为骨折修复的年轻化做出了贡献。 3)确定特定的巨噬细胞群及其分泌的蛋白质如何改变 骨折修复中的间充质分化。我们先前的工作表明，β-连环蛋白在 间充质细胞分化与骨折修复返老还童。在这里，我们将使用体外方法来 确定巨噬细胞的特定亚群及其分泌的蛋白质如何改变间充质 幼年和老年动物细胞中的细胞分化。最初的重点将是β-连环蛋白，但 也将使用不偏不倚的方法。 这项拟议的工作建立在我们之前关于骨折异慢性异生复壮的研究基础上。 修理。它将解决我们对导致复兴的机制的认识上的严重差距 异慢性异种共生驱动的表型。我们的工作还将确定一种新的治疗方法 解决老年患者的一个关键临床问题，即骨折愈合延迟。

The pace of bone repair slows with aging, increasing the chance of developing a delayed union or non-union. These complications are treated with surgical procedures causing significant morbidity and even mortality, especially in older adults. Here we will build on our previous work using heterochronic parabiosis (in which two mice of a different age share a blood supply) showing that exposure to a young circulation and young macrophage cells rejuvenates fracture repair in older mice. In our preliminary data we used cell lineage tracing analysis and parabiosis experiments to determine the developmental source of macrophage in fracture repair, and found these derived from a subpopulation of cells of yolk sac origin. Interestingly these cells reside in the spleen and are recruited through the circulation during bone repair. As mice age, this subpopulation of cells becomes depleted. In this proposal we study the role of this cell population and the factors they produce in the rejuvenation of fracture repair by undertaking the following aims: 1) Identify the role of macrophages derived from yolk sac progenitors in the rejuvenation of fracture repair. Heterochronic parabiosis in which these cells can be labeled or depleted will be investigated to define the contribution of young cells from this population of macrophage cells that can improve the quality of fracture repair in older animals. 2) Determine the function of genes expressed in unique macrophage subpopulations present in young mice in bone repair: We used single cell RNA sequencing and found a unique subpopulation of macrophages cells present in bone repair in only young animals. Mice lacking genes which encode for secreted proteins in various macrophage populations will be used in heterochronic parabiosis to determine their contribution to the rejuvenation of fracture repair. 3) Define how specific macrophage populations and the proteins they secrete alter mesenchymal differentiation in fracture repair. Our prior work showed an important role for beta-catenin in mesenchymal cell differentiation and in fracture repair rejuvenation. Here we will use in-vitro approaches to determine how specific subpopulations of macrophage cells and the proteins they secrete alter mesenchymal cell differentiation in cells from young and old animals. There will be an initial focus on beta-catenin, but an unbiased approach will be used as well. This proposed work builds on our prior studies of rejuvenation by heterochronic parabiosis in fracture repair. It will address critical gaps in our knowledge about the mechanism responsible for the rejuvenation phenotype driven by heterochronic parabiosis. Our work will also identify a novel therapeutic approach to address a critical clinical problem in older patients, delayed fracture healing.