Autophagy-Active Immune Cell Cluster and Crucial Gene Expression in Nonunion Via Single-Cell Sequencing and Functional Enrichment Analysis

Fracture nonunion represents a great challenge for orthopedic clinicians. This study aimed to identify autophagy-active immune cell clusters and key genes associated with nonunion progression using single-cell profiles and RNA-seq data. scRNA-seq profiles related to femoral nonunion were derived from the GEO database, and cell cluster annotation was subsequently performed. The AUCell package was utilized to calculate active cells based on autophagy gene expression, and cell communication was analyzed using CellChat. Twenty-one distinct cell clusters were identified from the nonunion tissues, which were annotated into 14 major cell types, and macrophages exhibited the highest AUCell score. Intercellular communication analysis revealed that the total number and intensity of overall cell interactions in the nonunion were notably increased compared with normal tissue. Furthermore, three ligand-receptor pathways (SPP1, MIF, and FN1) were notably altered between M1 macrophages and multiple other cell types, including endothelial cells, fibroblasts, pericytes, and NK cells. By combining the scRNA-seq and transcriptomic analyses, 21 key genes were identified, of which 12 genes, including CCL8, CDO1, and CEBPD, showed potential discriminatory ability for nonunion (AUC > 0.9). Moreover, the PPI network, ceRNA network, and mRNA-TF network were established to predict co-expression molecules. The results highlight the pivotal role of autophagy-active macrophages in nonunion and identify altered ligand-receptor pathways and 21 key DEGs associated with disease progression.