Dapi/phalloidin/wheat germ agglutin (WGA) and osteopontin staining

For the observation of the initial cell morphology of hMSCs seeded in composite scaffolds, the cells were fixed in 4% paraformaldehyde at room temperature. After washing in PBS, to block unspecific protein interactions, the samples were put in blocking solution (0.3% Triton X-100, 1% BSA, 10% donkey serum). Then, the samples were incubated with WGA-Alexa Fluor conjugate (Life Technologies) at 37°C for 1 h. Also, to observe the expression of osteopontin, one of the bone differentiation proteins, 10 μg mL⁻¹ osteopontin primary antibody (Abcam) was incubated overnight at 4°C. After washing, FITC-conjugated secondary antibody was incubated for 1 h at room temperature. Then, the samples were washed for nuclear labeling in the cells using mounting media (within DAPI, VECTOR) and observed using an LSM 5 Exciter confocal microscope (Carl Zeiss Microscopy GmbH, Olympus, Japan).