DLAT/DLD-dependent cuproptosis disrupts osteoblast-macrophage crosstalk through mtDNA-associated STING signaling under delayed-healing-like inflammatory-hypoxic conditions.

Impaired fracture repair is associated with sustained inflammation, mitochondrial dysfunction, and disrupted osteoblast-macrophage communication. Cuproptosis involves copper-dependent aggregation of lipoylated mitochondrial proteins and loss of iron-sulfur proteins, suggesting a potential connection between mitochondrial injury and osteoimmune dysfunction. This study investigated this relationship in an in vitro inflammatory-hypoxic model. Human hFOB 1.19 osteoblasts and THP-1-derived macrophages were studied under delayed-healing-like stress (DHS; 1% O₂ with TNF-α/IL-1β). Elesclomol-Cu (ES-Cu) served as a mechanistic probe, while tetrathiomolybdate (TTM) and DLAT/DLD depletion were used to investigate copper-dependent injury. Assessments included copper accumulation, cuproptosis-associated markers, mitochondrial function, mtDNA abundance, and STING-associated signaling. Conditioned-medium transfer and three-dimensional fibrin co-culture examined inflammatory responses and osteogenic differentiation. DNase treatment and macrophage STING depletion assessed contributions from extracellular DNA and STING. DHS increased intracellular copper (P = 0.0009) and produced modest cuproptosis-associated changes, which were more pronounced following ES-Cu addition. TTM or DLAT/DLD depletion attenuated injury, mitochondrial dysfunction, and mtDNA accumulation. Conditioned medium from injured osteoblasts increased macrophage CD86 positivity and inflammatory cytokine secretion while reducing CD206 positivity. DNase treatment or macrophage STING depletion attenuated inflammatory responses. Macrophage-conditioned medium subsequently suppressed ALP activity, mineralization, and osteogenic gene expression in recipient osteoblasts. These endpoints improved following donor-cell interventions or macrophage STING depletion. Three-dimensional co-culture showed corresponding changes. The findings support a model linking experimentally induced copper-dependent osteoblast injury under DHS with mtDNA-associated STING signaling, macrophage inflammatory responses, and impaired osteogenic differentiation. They do not establish mtDNA-specific cGAS activation or cuproptosis-specific effects of DLAT/DLD depletion, and residual treatment carryover remains unresolved. Validation beyond these cell-line systems is required before extending the findings to clinical delayed fracture healing.