Abstract
The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age‐related diseases. Therefore, we investigated age‐related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA‐seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up‐regulation of M1/pro‐inflammatory genes in macrophages from old mice as well as dysregulation of other immune‐related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age‐matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA‐seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age‐related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.
Highlights
Fracture healing follows a distinct temporal sequence characterized by an initial inflammatory phase followed by anabolic and catabolic phases (Little, Ramachandran, & Schindeler, 2007).The inflammatory phase is characterized by recruitment of innate and adaptive immune cells to the fracture site (Hankenson, Zimmerman, & Marcucio, 2014)
We have shown the importance of macrophages in fracture healing in previous work, where young adult mice lacking the C-C Motif Chemokine Receptor 2 (Ccr2) demonstrate significantly reduced trafficking of macrophages to the fracture callus and a resulting delay in fracture healing (Xing, Lu, Hu, Miclau, et al, 2010; Xing, Lu, Hu, Yu, et al, 2010)
We demonstrated that a pharmacologic (PLX3397) leading to a decrease in macrophages recruitment to the fracture site of old mice improves fracture healing outcomes
Summary
Fracture healing follows a distinct temporal sequence characterized by an initial inflammatory phase followed by anabolic and catabolic phases (Little, Ramachandran, & Schindeler, 2007). To further assess differences between old and young macrophages, samples were hierarchically sorted based on their differential expression of 14 genes associated with characteristic macrophage cytokines and markers of M1 and M2 macrophages This analysis demonstrated that mice sort by age and that old mice have increased expression of pro-inflammatory cytokines and markers of M1 macrophages (Figure 3d). RNA-seq analysis demonstrated that only 64 genes were significantly differentially expressed in old macrophages from mice treated with PLX3397 compared to young macrophages; genes were up-regulated and genes were down-regulated more than twofold (Figure 5b). Principal component analysis further demonstrates that the macrophages from old mice treated with PLX3397 cluster closely with the young mice compared to the old with less transcriptomic heterogeneity (Figure 5d) This suggests that the inflammatory macrophages that are recruited to the bone fracture are substantially different between old and young mice, but the remaining tissue-resident macrophages demonstrate less age-related changes
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