Morphological and Cytotoxic Effects of Metal Salts on Osteoblast Cells in Three-Dimensional Spheroidal Cultures

The skeleton provides structural support, organ protection, and mineral storage, making the long-term biocompatibility of orthopedic implants essential. Although stainless steel and cobalt-based alloys are widely used in these devices, corrosion can release metal ions that induce inflammatory and cytotoxic responses in surrounding bone tissue. Because most in vitro studies rely on conventional two-dimensional (2D) cultures, three-dimensional (3D) spheroids offer a more physiologically relevant model for evaluating metal ion toxicity. In this study, human fetal osteoblast (hFOB) spheroids were exposed to CrCl₃, CoCl₂, or NiCl₂ at 100 or 500 μM for 12 days. Morphological alterations were evaluated by scanning electron microscopy and histology, while cytotoxicity and apoptosis were assessed using resazurin, live/dead fluorescence, and annexin V/propidium iodide staining. CrCl₃ caused minimal effects at 100 μM and only moderate cytotoxicity at 500 μM. In contrast, CoCl₂ and especially NiCl₂ significantly reduced spheroid growth, induced retraction of cytoplasmic extensions, membrane damage, and increased apoptosis, with the most severe effects observed at 500 μM. Fluorescence and Annexin V staining confirmed a higher abundance of apoptotic bodies after CoCl₂ and NiCl₂ exposure than after CrCl₃ treatment. These findings demonstrate that 3D osteo-spheroids provide a physiologically relevant platform for investigating the biological effects of metal ions released from orthopedic implants and reveal that nickel and cobalt ions exert substantially greater cytotoxicity than chromium under prolonged exposure conditions.