Real-Time Fluorescence Visualization of the Dynamic Distribution of Zn²⁺ Ions during Osteoblast Differentiation

The differentiation and maturation of osteoblasts are essential for bone formation. Zn²⁺ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn²⁺ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn²⁺ within the nucleus. Here, a novel Zn²⁺ fluorescent probe (NTAD-N1) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn²⁺ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn²⁺ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn²⁺ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn²⁺ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn²⁺ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.