Model 700

To evaluate cellular cytoskeletal organization on the pristine PLGA, nHA/PLGA, and BMP-g-nHA/PLGA hybrid nanofiber scaffolds, double staining was performed according to the previously reported method [22 (link)]. Briefly, osteoblast cells were seeded onto the nanofiber scaffolds (2 × 10⁴ cells/mL) separately and cultured for 3 days. The cells were then fixed with 4% paraformaldehyde in PBS. After fixation, the samples were washed using a PBS containing 0.05% Tween-20. The samples were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 25°C and then incubated for 30 min in a PBS containing 1% bovine serum albumin (BSA). This was followed by the addition of 5(6)-tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (TRITC Millipore) for approximately 1 h. The nanofiber scaffolds were washed three times (10 min each) using the buffer solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI, Millipore) for 5 min. Fluorescence images were visualized using a confocal laser-scanning microscope (Model 700; Carl Zeiss, Oberkochen, Germany).