Single-cell RNA-seq data reveals a critical role of pro-inflammatory macrophage and fibroblast cells in bone marrow environment after bone fracture

Abstract Single-cell RNA sequencing ("scRNA-Seq") examines the cell population at the single-cell level. The single cell changes in the osteoimmunological microenvironment in fresh and old fractures have not been studied. We used single cell transcriptomics in this study to uncover differences in the molecular composition and cellular signaling in bone tissue from fresh and old fractures.We first searched for and downloaded single-cell omics data from the GEO database, which included both fresh and old fracture samples from patients. After applying UMI detection, reducing the dimensions, and conducting principal component analysis, we visualized the data with tSNE and UMAP and identified the marker genes of the cell subsets. The differences of the differentially expressed genes and the signalling pathways of the cell-cell interaction between the two groups of samples were compared by means of Findmarkers and cellchat.The microenvironment in fracture tissue was analysed using a cell characterisation map, resulting in the identification of 18 distinct cell subsets, comprising of macrophages, fibroblasts, B cells, T cells, neutrophils and plasma cells. In comparison to fresh fractures, there was a significant increase in the number of macrophages in the old fracture samples. The number of fibroblasts was not significantly changed. The results of differential expression gene analysis showed that fibroblasts in old fractures were mainly enriched in immune, inflammatory and neutrophil degranulation reactions. TXNIP expression was significantly upregulated. Macrophages were mainly enriched in inflammatory response, immune response, antigen presentation response and cell migration signalling pathways. Among them, AREG was significantly upregulated in old fractures. In old fractures, the interaction between macrophages and other cells was significantly increased. Macrophages regulate other cells mainly through the ANXA1-FRP1 signalling pathway, thereby influencing the formation of callus and the healing of the fracture. Our findings uncovered that fibroblasts regulate inflammation and immune response via the TXNIP pathway. Macrophages influence fracture healing by changing their population and interacting with other cells via the ANXA1-FRP1 pathway.