Real-Time Fluorescence\nVisualization of the Dynamic\nDistribution of Zn<sup>2+</sup> Ions during Osteoblast Differentiation

The differentiation and maturation of osteoblasts are\nessential\nfor bone formation. Zn<sup>2+</sup> plays a crucial role in cell differentiation\nand is involved in osteogenic differentiation. The concentration and\ndistribution of Zn<sup>2+</sup> in the nucleus and cytoplasm indicate\nthe differentiation states of osteoblasts. However, there is an absence\nof a real-time method for monitoring the dynamic fluctuations of endogenous\nZn<sup>2+</sup> within the nucleus. Here, a novel Zn<sup>2+</sup> fluorescent\nprobe (<b>NTAD-N1</b>) with nuclear membrane permeability was\ndesigned and developed, allowing for distribution throughout the entire\ncell, including the nucleus. The <b>NTAD-N1</b> probe successfully\nshowed the dynamic distribution and concentration changes of Zn<sup>2+</sup> in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during\nthe 21-day differentiation period. The results showed that free Zn<sup>2+</sup> increased significantly during differentiation of osteoblasts\n(2–21 days). Importantly, after 4 days of differentiation,\nosteoblasts are mainly distributed in the nucleus, which is confirmed\nby metallothionein expression. Subsequently, the level of free Zn<sup>2+</sup> in the cytoplasm remained at a high level, which promoted\nthe increase in alkaline phosphatase activity and inhibited the activity\nof cis-aconitase in the tricarboxylic acid cycle, resulting in the\naccumulation of citric acid. This series of events promotes the formation\nof mineralized nodules. In the process of osteoblast differentiation,\nthe detection time of Zn<sup>2+</sup> (≤7 days) is ahead of\nthe late marker of alkaline phosphatase (14 days) and mineralized\nnodules (14–21 days). This indicates that Zn<sup>2+</sup> can\nbe used as a biomarker and an intervention point for early differentiation\nof osteoblasts.