Temporal Molecular Signatures of Early Human Clavicle Fracture Healing: Characterization of Hematological, Cytokine, and miRNA Profiles

Fracture healing failure affects millions globally, yet early molecular mechanisms remain poorly understood. This study aimed to characterize initial fracture response through analyzing peripheral blood hematology, multiplex cytokine profiles, and microRNA (miRNA) expression in fracture hematoma within the first 5 days post-injury. In a prospective cohort of 64 patients with acute clavicle fractures, we assessed hematological parameters, cytokine levels via multiplex immunoassays, and miRNA expression through RNA sequencing, and quantitative PCR (qPCR) validation. Fracture severity and time elapsed post-injury were key drivers of molecular response variability. Severe fractures (type C) were associated with older patient age and impaired hematological parameters, including reduced hemoglobin, erythrocyte counts, and hematocrit. Leukocyte counts declined over time, reflecting evolving systemic inflammation. Severity-dependent cytokines included eotaxin, interferon alpha-2 (IFNα2), interleukin-1 alpha (IL-1α), macrophage inflammatory protein-1 (MIP-1α), whereas interferon gamma-induced protein 10 (IP-10) and MIP-1α distinguished temporal healing phases. MiRNA profiling revealed 55 miRNAs with significant time-dependent expression changes (27 downregulated, 28 upregulated). Five key miRNAs (miR-140-5p, miR-181a-5p, miR-214-3p, miR-23a-3p, miR-98-5p) showed robust temporal patterns and enrichment in cytokine signaling pathways critical for bone repair. This work presents the first detailed molecular portrait of early human fracture healing, highlighting hematological, immune cytokine, and miRNA networks orchestrating repair. These insights provide a foundation for biomarkers development to predict healing outcomes and support precision-targeted interventions in fracture management.