Analysis of osteo-immune crosstalk during bone healing by longitudinal intravital imaging

骨折愈合始于炎症阶段，在此期间免疫细胞侵入受损的组织区域。它之后是一个阶段的骨修复。这两个过程都涉及细胞和亚细胞水平上的高度动态过程，如免疫细胞迁移以及免疫和间质之间的细胞相互作用。活体显微镜是研究这些过程的首选方法，然而，到目前为止，纵向分析骨再生的时空方面是不可能的。为了克服这一挑战，我们在第一个资助期开发了一种显微内窥镜设备(肢体)，允许通过对骨髓中相同组织区域的重复成像来分析体内骨愈合过程中的骨免疫相互作用。在第二个资助期，我们的目标是通过分析不同的小鼠骨折愈合模型中衰老对基质和免疫隔室的作用，来剖析衰老对基质和免疫隔室的作用。免疫细胞衍生因子的影响将使用条件基因敲除小鼠品系进行测试。免疫细胞和间充质细胞的分化将通过显微内窥镜荧光寿命成像对细胞代谢的分析来监测。已知的血管生成在空间和机械上与骨形成有关，将使用肢体分析不同年龄组的血管生成。

Fracture healing is initiated with an inflammatory phase, during which immune cells invade the damaged tissue area. It is followed by a phase of bone repair. Both processes involve highly dynamic processes on the cellular and subcellular level, such as immune cell migration and cellular interactions between immune and stromal compartments. Intravital microscopy is the method of choice to study these processes, however, analyzing the spatiotemporal aspects of bone regeneration longitudinally has not been possible until now. To overcome this challenge, we have developed a microendoscopic device (LIMB) in the first funding period, allowing to analyze osteo-immune interactions during bone healing in vivo by repeated imaging of the same tissue region in the bone marrow. In the second funding period, we aim to use LIMB to dissect the role of aging on stromal and immune compartments by analyzing their dynamics in different mouse models of fracture healing. The impact of immune cell-derived factors will be tested using conditional knockout mouse strains. Differentiation of immune and mesenchymal cells will be monitored via the analysis of cellular metabolism by microendoscopic fluorescence lifetime imaging. Angiogenesis, which is known to be spatially and mechanistically linked to bone formation, will be analyzed in different age cohorts using LIMB.