The role of macrophage subpopulations in the rejuvenation of fracture repair

骨修复的速度随着年龄的增长而减慢，增加了延迟愈合或不愈合的机会。 这些并发症通过外科手术治疗，导致显著的发病率甚至死亡率， 尤其是老年人。在这里，我们将建立在我们以前的工作，使用异时共生（其中两个 不同年龄的小鼠共享血液供应），显示暴露于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， 分析和共生实验以确定骨折修复中巨噬细胞的发育来源， 并发现这些细胞来自卵黄囊起源的细胞亚群。有趣的是，这些细胞位于 脾脏，并在骨修复过程中通过循环招募。随着小鼠年龄的增长， 变得枯竭。在这个建议中，我们研究了这个细胞群的作用以及它们在细胞周期中产生的因子。 通过实现以下目标使骨折修复恢复活力： 1)鉴定来自卵黄囊祖细胞的巨噬细胞在胚胎再生中的作用 骨折修复异时共生，其中这些细胞可以被标记或耗尽将进行研究 为了确定年轻细胞的贡献，从这个群体的巨噬细胞，可以提高质量 在老年动物中的骨折修复。 2)确定存在于巨噬细胞中的独特巨噬细胞亚群中表达的基因的功能， 年轻小鼠的骨修复：我们使用单细胞RNA测序，发现了一个独特的亚群， 巨噬细胞仅存在于年轻动物的骨修复中。小鼠缺乏编码 各种巨噬细胞群体中的分泌蛋白将用于异时联体，以确定 他们对骨折修复的复兴的贡献。 3)定义特定的巨噬细胞群体及其分泌的蛋白质如何改变 间充质分化在骨折修复中的作用我们先前的工作表明β-连环蛋白在 间充质细胞分化和骨折修复复壮。在这里，我们将使用体外方法， 确定巨噬细胞的特定亚群及其分泌的蛋白质如何改变间质细胞的功能， 年轻和年老动物的细胞分化。将有一个最初的重点β-连环蛋白，但 也将使用无偏见的方法。 这项工作是建立在我们以前的研究复兴异时联体在骨折 修复.它将解决我们对振兴机制的认识方面的关键空白 由异时共生驱动的表型。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，延迟骨折愈合。

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.