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INTRODUCTION: The pace of bone healing slows with age, increasing the chance of developing a delayed union and malunion. Circulating monocytes and tissue-resident macrophages are mononuclear phagocytes that are increasingly recognized as an essential player of bone repair and regeneration. Once the bone is damaged, an initial inflammatory response is induced by immune cells such as monocyte/ macrophage and neutrophil. They release factors that promote the recruitment, proliferation, and activation of a variety of hematopoietic and mesenchymal lineage cells that orchestrate tissue repair. However, the ablation of monocyte/ macrophage during the bone healing lead to the pathological fibrosis and decrease the number of mesenchymal progenitor cells resulting in impaired normal bone regeneration. Recent data suggest that circulating macrophage cells are at least partially responsible for regulating the age-related pace of repair. Here we used single-cell RNA sequencing (scRNA-seq) to identify the diversity and trajectories of macrophage cells during bone healing, and compared these trajectories between young and old mice. METHODS: 2-month-old (young) and 20-month-old (old) C57BL6 mice underwent drill hole surgery on the right femur. A 0.8 mm hole was made in both cortexes of the femur. Mice were sacrificed at 3, 7, and 21 days after surgery and bone marrow cells at the injury site were collected and submitted to the scRNA-seq experiment. Uninjured mice were used as Day 0 control. Two mice in each time point and a total of 8 mice in each group were subjected to the scRNA-seq experiments. All individual cell was encapsulated in one droplet with 10X Genomics Gem Code Technology. Data clustering was performed with Surat and Monocle2 R package. In addition to the young and old wild-type mice, 8 week-old Cathelicidin antimicrobial peptide (Camp) knockout mice (Camp-/-) were subjected to the histological and micro CT analysis. RESULTS: t-distributed Stochastic Neighbor Embedding (tSNE) plot of entire data set based on transcriptional similarity visualised the diversity and heterogeneity of about 70K bone marrow cell during bone healing in both young and old mice (Figure1 a). We selected the cells in macrophage and monocyte clusters and performed a second round subclustering analysis. Subclustering tSNE revealed 13 subpopulations of macrophage/ monocyte subset and clearly showed young mice specific subpopulation at Day 7 (Figure 1b, Gray circle). In this young mouse-specific subpopulation, 98 differentially expressed genes were clarified. To identify multi-state transition of macrophage during bone healing, all cells from macrophage/ monocyte cluster in young mice were assigned into the pseudotime-trajectory analysis using Monocle2. Monocle2 automatically identified 3 branches (sate 0, 1, and 2) and one branch point with significant differential expression across the time axis (Figure 2a). A global search for genes with significant branch-dependent expression using branch expression analysis modeling (BEAM) discovered differentially expressed genes in state 1 and 2 (Figure 2b). Computational BEAM also revealed unique gene expression patterns of macrophage along the pseudotime trajectories. Camp was a one candidate gene upregulated in young mice at day 3 (in state 2), and we confirmed the protein expression of CAMP in the drill hole site of the young femur (Figure 2c). To examine the contribution of the Camp protein product to the bone healing, wild-type and Camp-/- mice underwent drill hole surgery and the micro CT analysis. The data clearly showed that ablation of Camp delayed the femoral bone healing as compared to young wild-type mice, similar to the old mice phenotype (Figure 3). DISCUSSION: Single-cell genomics enabled the identification of the transcriptional regulation of complex biological processes and highly heterogeneous cell populations. The scRNA-seq data revealed a landscape of macrophage subpopulation in young and aged mice during the bone healing and identified a specific macrophage subpopulation at Day 7 in young mice that was not present in old mouse. Camp is known as a multifunctional innate immune effector that can attract and activate inflammatory cells, including dendritic cells and monocytes. Impaired inflammatory cascade due to the Camp ablation could contribute to the delayed bone healing. Our data is consistent with the notion that specific macrophage cell populations are orchestrating bone repair, and that CAMP could be a therapeutic target to regulate the pace of fracture repair. SIGNIFICANCE: Single cell RNA sequencing reveals the diversity and unique trajectories of macrophage during the bone healing in young and old mice. Depleting a gene expressed in the unique subpopulation slowed bone healing. Acknowledgement: This research is supported by a grant from the NIH (R01AG049745)
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