The optimized electrospinning conditions used in the present study were tip-to-collector distance 20 cm, applied voltage 20 kV, needle diameter 20 G (0.9 mm), and flow rate 1 mL/h. The electrospun nanofibers collected were removed from the collector and dried overnight at 40°C to remove the remaining solvent. After drying, the sample was sputter-coated with gold and its morphology was observed by field emission scanning electron microscopy (FESEM; 400 Hitachi, Tokyo, Japan). The same procedure was adapted for the preparation
of the electrospun PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds. Briefly, both pristine nHA and insulin-grafted nHA-I selleckchem were added into the PLGA polymer solution and were mechanically dispersed via alternate stirring and
sonication. After dispersion, the PLX3397 samples were subjected to electrospinning process. Osteoblastic cell culture To examine the interaction of the PLGA/nHA-I and PLGA/nHA composite nanofiber scaffolds with osteoblastic cells (MC3T3-E1), the composite nanofiber scaffolds were Selleck P005091 cut into small circular discs, fitted inside a 4-well culture dish, and immersed in MEM medium containing 10% FBS (Gibco; Invitrogen, Carlsbad, CA, USA). Subsequently, 1 mL of the MC3T3-E1 cell solution (3 × 104 cells/mL) was added to the surface of the composite nanofiber scaffolds and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 1 and 3 days. After incubation, the supernatant was removed and the composite nanofiber scaffolds were washed twice with phosphate-buffered saline (PBS; Gibco, Langley, OK, USA) and fixed in a 2.5% glutaraldehyde solution for 15 min. The samples were
then dehydrated, dried in a critical point drier, and sputter-coated with gold. The surface morphology of the composite nanofiber scaffolds was observed by FESEM (400 Hitachi; Tokyo, Japan). Cytoskeletal organization To evaluate the cytoskeletal organization of cells onto the PLGA/nHA-I and PLGA/nHA composite as well as pristine PLGA nanofiber scaffolds, double staining was performed according to the manufacturer’s protocol. Briefly, osteoblast cells were seeded onto the scaffolds (2 × 104 RG7420 research buy cells/mL) and were cultured for 3 days. The cells were fixed with 4% paraformaldehyde in PBS. After fixation, the samples were washed using PBS buffer solution 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 PBS containing 1% bovine serum albumin (BSA). This was followed by the addition of 5(6)-tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (Millipore) (TRITC) for approximately 1 h. The samples were washed three times (10 min each) using the buffer solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Millipore) for 5 min.