*2.7. ALP Activity and Bone Mineralization Assay*

ALP activity can provide a useful index of the osteoblastic phenotype. Thus, the osteoblastic differentiation of MC3T3-E1 cells was assessed by measuring the ALP activity with an ALP assay kit (DALP-250, BioAssay Systems, USA) at periods of 3, 5, 7, and 14 days after cell seeding. The composite nanofibers seeded with cells washed with DPBS and incubated in 0.5 mL of DW containing 0.2% Triton X-100. The cell lysates were mixed with *p*-nitrophenyl phosphate solution. In the presence of ALP, *p*-nitrophenyl phosphate can be hydrolyzed to *p*-nitrophenol and the rate of *p*-nitrophenol production is proportional to the ALP activity. Therefore, the level of *p*-nitrophenol production was determined by measuring the absorbance at 405 nm and normalized to 1 × <sup>10</sup><sup>4</sup> cells.

Bone mineralization capability of the composite nanofibers was assayed by the alizarin red S (ARS) staining method. ARS can selectively bind to calcium ions and thus calcium deposition is easily measured by the use of ARS. After 3, 5, 7, and 14 days of cell culture on the different nanofibers, the cells were fixed with 4% glutaraldehyde for 30 min and then stained with 1 mL of 40 mM ARS (pH 4.1). After incubation for 20 min, the specimens were washed thrice with DW to remove unreacted ARS and dissolved in 1 mL of 10% cetylpyridinium chloride. The absorbance of the supernatant at 540 nm was obtained using microplate reader and was normalized to 1 × <sup>10</sup><sup>4</sup> cells.
