*3.5. ALP Activity and Bone Mineralization*

ALP catalyzes the cleavage of organic phosphate esters and plays an important role during the formation process of bone nodule [1,2]. ALP activity is considered as an early marker of osteoblastic differentiation because it significantly increases during the differentiation step. Therefore, we examined ALP activity to evaluate the capability of the PVDF composite nanofibers for promoting osteoblastic differentiation. As a result, the cells cultured on PE06 exhibited higher ALP activity than those cultured on the other composite nanofibers at all time points (Figure 10a). Similar results have been previously reported, in that various flavonoids—such as green tea catechins, quercetin, and kaempferol—significantly increased ALP activity by the activation of the extracellular signal-regulated kinase pathway and endoplasmic reticulum binding [40–42].

**Figure 10.** (**a**) ALP activity and (**b**) quantitative calcium deposition of MC3T3-E1 cells on the PVDF composite nanofibers after culturing for 3, 5, 7, and 14 days (*n* = 6). The values of ALP activity and quantitative calcium deposition were normalized by the cell number (1 <sup>×</sup> 104 cells). Significant difference from the pure PVDF nanofiber at each time point was denoted as *p*\* < 0.05.

The mineralized bone nodules are usually formed via three main stages such as cell proliferation, ECM development, and mineralization. Therefore, the osteogenesis capability of the cells as a late-stage marker for osteogenic differentiation can be estimated by the determination of bone-like calcium deposits produced on different PVDF composite nanofibers using ARS staining [1,4]. After the incubation for 3, 5, 7, and 14 days, the absorbance changes of ARS in the red-stained composite nanofibers were used to quantify the calcium mineralization. Figure 10b exhibits the absorbance of ARS extracted from the stained calcium deposits in different composite nanofibers. The increase of calcium deposition on the composite nanofibers with time was observed in all samples. Among the samples, PE06 exhibited the highest calcium deposition compared to the other samples at all time points caused by increased osteogenesis capability [42]. These results indicated that the amount of

POSS–EGCG conjugate present in the composite nanofibers significantly influenced the bone-forming ability of the MC3T3-E1 cells.
