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Severe inflammation and myogenic differentiation disorder are the major obstacles to skeletal muscle healing after injury. MicroRNAs (miRNAs) play an important role as regulatory molecules during the process of muscle healing, but the detailed mechanism of miRNA‐mediated intercellular communication between myoblasts and macrophages remains unclear. Here, it is reported that myoblasts secrete miRNAs‐enriched exosomes in the inflammatory environment, through which miR‐224 is transferred into macrophages to inhibit M2 polarization. Further data demonstrate that WNT‐9a may be a direct target of miR‐224 for macrophage polarization. In turn, the secretome of M1 macrophages impairs myogenic differentiation and promotes proliferation. Single‐cell integration analysis suggests that the elevation of exosome‐derived miR‐224 is caused by the activation of the key factor E2F1 in myoblasts and demonstrates the RB/E2F1/miR‐224/WNT‐9a axis. In vivo results show that treatment with antagomir‐224 or liposomes containing miR‐224 inhibitors suppresses fibrosis and improves muscle recovery. These findings indicate the importance of the crosstalk between myoblasts and macrophages via miRNA‐containing exosomes in the regulation of macrophage polarization and myogenic differentiation/proliferation during muscle healing. This study provides a strategy for treating muscle injury through designing an M2 polarization‐enabling anti‐inflammatory and miRNA‐based bioactive material.

Introduction. Regeneration of fibrochondrocytes is essential for the healing of the tendon-bone interface (TBI), which is similar to the formation of neurogenic heterotopic ossification (HO). Through single-cell integrative analysis, this study explored the homogeneity of HO cells and fibrochondrocytes. Methods. This study integrated six datasets, namely, GSE94683, GSE144306, GSE168153, GSE138515, GSE102929, and GSE110993. The differentiation trajectory and key transcription factors (TFs) for HO occurrence were systematically analyzed by integrating single-cell RNA (scRNA) sequencing, bulk RNA sequencing, and assay of transposase accessible chromatin seq. The differential expression and enrichment pathways of TFs in heterotopically ossified tissues were identified. Results. HO that mimicked pathological cells was classified into HO1 and HO2 cell subsets. Results of the pseudo-temporal sequence analysis suggested that HO2 is a differentiated precursor cell of HO1. The analysis of integrated scRNA data revealed that ectopically ossified cells have similar transcriptional characteristics to cells in the fibrocartilaginous zone of tendons. The modified SCENIC method was used to identify specific transcriptional regulators associated with ectopic ossification. Xbp1 was defined as a common key transcriptional regulator of ectopically ossified tissues and the fibrocartilaginous zone of tendons. Subsequently, the CellPhoneDB database was completed for the cellular ligand-receptor analysis. With further pathway screening, this study is the first to propose that Xbp1 may upregulate the Notch signaling pathway through Jag1 transcription. Twenty-four microRNAs were screened and were found to be potentially associated with upregulation of XBP1 expression after acute ischemic stroke. Conclusion. A systematic analysis of the differentiation landscape and cellular homogeneity facilitated a molecular understanding of the phenotypic similarities between cells in the fibrocartilaginous region of tendon and HO cells. Furthermore, by identifying Xbp1 as a hub regulator and by conducting a ligand–receptor analysis, we propose a potential Xbp1/Jag1/Notch signaling pathway.