Abstract
Zinc oxide nanoparticles (ZnO-NPs) have attracted considerable interest for biomedical applications due to their favorable physicochemical and biological properties; however, their biological effects are highly concentration-dependent, and optimal conditions for osteogenic effects remain unclear. In this study, we aimed to systematically evaluate the concentration-dependent effects of ZnO-NP extracts on osteogenic responses in pre-osteoblastic cells. ZnO-NPs were first characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD), and extracts were prepared at concentrations of 0.01, 0.1, 1, and 10 mg/mL (Zn0.01, Zn0.1, Zn1, and Zn10), respectively. MC3T3 - E1 cells were then exposed to these extracts to assess cytocompatibility and osteogenic activity. Cytocompatibility was assessed using WST-1 cell viability assays and Live/Dead assays, while Zn2+ ion release was quantified by inductively coupled plasma mass spectrometry (ICP-MS). Osteogenic activities were performed using quantitative polymerase chain reaction (qPCR) analysis of osteogenic markers, alkaline phosphatase (ALP) activity assay at 7 and 14 days, and Alizarin Red S (ARS) staining for biomineralization up to 21 days. Among the groups, the Zn0.01 group significantly enhanced cell proliferation and viability, whereas the Zn10 group induced marked cytotoxicity. Notably, the Zn0.01 group exhibited the highest upregulation of osteogenic genes, including Runx2, Ocn, and Bsp, together with increased ALP activity and biomineralization. These findings demonstrate that ZnO-NPs exert concentration-dependent biological effects and suggest that low concentrations may support osteogenic responses while maintaining cytocompatibility. This study provides a framework for optimizing the use of ZnO-NPs in hard tissue-related regenerative applications.