Single-Cell Re-Mining Identifies a Pathogenic Fibroblast Subpopulation and Nominates Exploratory Therapeutic Hypotheses in Human Bone Nonunion

Bone nonunion is a significant clinical problem, yet the cellular mechanisms that sustain failed repair in humans remain poorly understood. Here, we re-analyzed a human nonunion single-cell RNA sequencing (scRNA-seq) dataset to move beyond broad descriptive cell-population changes and generate a subtype-resolved, hypothesis-oriented framework. Across 16 major lineages, differential abundance testing revealed robust expansion of fibroblast, chondrocyte, and macrophage neighborhoods in nonunion. Intercellular communication reconstruction further showed that macrophages were the dominant upregulated signal “senders”, whereas fibroblasts were the primary “receivers”, with a disease-enhanced macrophage-to-fibroblast TGFB1–(TGFBR1+TGFBR2) axis forming a central hub. Fibroblasts in nonunion also showed metabolic rewiring and an increased senescence signature, including strengthened metabolite-mediated macrophage-to-fibroblast signaling (e.g., glutamine-linked pathways). Further subclustering identified a disease-enriched fibroblast “Fib1 executor” state that acts as a pro-fibrotic signaling sink, governed by a 13-transcription-factor regulon and a compact five-gene effector module (ACTA2, F3, THBS1, SERPINE1, NTM). Finally, we performed an exploratory computational compound-prioritization analysis linked to the Fib1-centered program, generating preliminary hypotheses for future validation rather than validated therapeutic nominations. Together, our results suggest that human nonunion contains a disease-enriched macrophage-to-fibroblast communication program and a fibroblast-centered effector signature that may help guide future mechanistic and therapeutic studies.