The reduction of Alamar blue was measured at 560 nm (reference wavelength 610 nm) after 5-h incubation at 37 °C using a microplate reader (Infinite 200, Tecan).

Generation of BMP2 expressing C2C12 cells

C2C12 cells were electroporated together with 0.4 μg of a BMP-2 expression vector (a kind gift from Dr. Katagiri) and 0.1 μg of pCMV-Tag, which expresses a neomycin-resistance gene. Culture medium was supplemented with 300 μg/ml of G418 (Fisher, Pittsburgh, PA, USA) for 9 days. Clones were picked, amplified, and screened by alkaline phosphatase (ALPL; a downstream target for BMP-2 signaling) staining.

Zymography and Western blotting

Protein samples from the transfected cells were prepared in 1× SDS gel-loading buffer (Laemmli buffer) without adding β-mercaptoethanol and quantified using the Pierce™ Coomassie Plus Assay kit (Thermo Scientific, Rockford, IL, USA). Without heat denaturation, equal amount of protein samples (50 μg) were loaded on a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was incubated in NBT/BCIP (Roche, Mannheim, Germany) staining solution until the bands corresponding to ALPL were clearly visible. Same protein extracts upon heat denaturation in the presence of β-mercaptoethanol were used for Western blotting. The primary antibody used for the analysis was anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). An anti-actin antibody raised in rabbit anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). The secondary antibody was anti-rabbit HRP-IgG (Cell Signaling Technology, Beverly, MA, USA).

Cell proliferation

Nuclear staining was done by H33258 (Sigma-Aldrich, Saint Louis, MO, USA). After washing in PBS, cells grown on the implants were imaged using an inverted fluorescent microscope (EVOS FL, Thermo Fisher Scientific) and cell nuclei were counted.