Annexin V‐Functionalized Injectable Calcium‐Deficient Hydroxyapatite for Extracellular Vesicles Binding and Controlled Release

ABSTRACT Calcium phosphates alone have a limited induction potential in challenging bone conditions, necessitating strategies that enhance cellular responses and matrix‐related processes. An injectable formulation combining calcium‐deficient hydroxyapatite (CDHA) with mesenchymal stem cell‐derived extracellular vesicles may present a promising approach for modulating osteogenic activity. A dual formulation composed of calcium‐deficient HA and human bone marrow mesenchymal stem cell‐derived extracellular vesicles, incorporating Annexin V as a binding molecule, was generated and validated. Structural and molecular characterization was performed using Raman spectroscopy, electron paramagnetic resonance, and sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Real‐time impedance‐based cell proliferation analysis was applied to determine biologically effective concentrations. The effects of the formulation on human osteoblast behavior and early osteogenic activity were additionally evaluated. Raman spectroscopy confirmed structural integration within the formulation termed CHAnEx, showing peak shifts and spectral features at 959, 630, and 806 cm −1 , indicating molecular interactions between calcium‐deficient HA, Annexin V, and extracellular vesicles. Electron paramagnetic resonance (EPR) analysis revealed distinct g‐factor shifts consistent with binding interactions. Real‐time proliferation analysis identified biologically effective concentrations of calcium‐deficient HA and extracellular vesicles using human osteoblast models. The combined formulation increased alkaline phosphatase activity, showing approximately a 20%–25% increase compared to control groups. These findings indicate that the calcium‐deficient HA‐extracellular vesicle formulation supports functional interactions and modulates osteoblast responses under in vitro conditions, suggesting its relevance as an injectable biomaterial platform.