Osteogenic Differentiation Restores Physiological Mineralization Pathways in SaOS-2 Osteosarcoma Cells

Osteosarcoma, the most common primary malignant bone tumor, is characterized by disruptions in osteogenic differentiation and matrix mineralization. In physiological bone formation, mesenchymal stem cells differentiate into osteoblasts and coordinate the deposition of hydroxyapatite, the principal mineral component of bone. While bone mineralization has long been considered an extracellular process, increasing evidence indicates that early mineral formation is initiated intracellularly through calcium accumulation and precursor mineral formation within specific mitochondria. However, the organization and regulation of this intracellular pathway remain poorly understood, particularly in pathological contexts such as osteosarcoma. This study aimed to investigate whether osteogenic induction can restore physiological biomineralization in SaOS-2 osteosarcoma cells by synchrotron-based cryogenic imaging and spectroscopy. Mineralization dynamics were compared with induced human bone marrow mesenchymal stem cells as a non-pathological reference model of osteogenic differentiation and with ex vivo human bone tissue. A coordinated intracellular biomineralization pathway was identified in which calcium-rich precursors accumulate within mitochondria, are transferred through vesicular trafficking, and progressively mature into hydroxyapatite during osteogenic differentiation, prior to extracellular matrix mineralization. Under osteogenic induction, SaOS-2 cells recapitulate key features of osteogenesis, producing hydroxyapatite chemically consistent with mature human bone. In contrast, in the absence of induction, mineralization remains incomplete and chemically heterogeneous. These findings link differentiation to mineral chemistry and trafficking, providing a framework to resolve early biomineralization defects in bone cancer and suggesting differentiation-based treatments as a complementary therapeutic avenue for osteosarcoma. STATEMENT OF SIGNIFICANCE: Osteosarcoma is an aggressive bone cancer characterized by a defective differentiation, that leads to an abnormal mineral formation. Using advanced imaging and X-ray spectroscopy techniques, we provide experimental evidence demonstrating how the induction of osteogenic differentiation reprograms human malignant cells, reactivating a coordinated biomineralization pathway culminating in the formation of mature hydroxyapatite chemically indistinguishable from that of native human bone. These findings provide evidence that early human bone formation involves intracellular mechanisms that can be restored in cancer cells. Overall, this work advances the understanding of biomineralization process and supports differentiation-based strategies for developing targeted bone cancer treatments.