Alkaline phosphatase immunostaining and assay

BMP-2-transfected C2C12 cells were fixed in 4% PFA for 15 min, and then blocked with 5% bovine serum albumin (Fisher, Pittsburgh, PA, USA) in Tris buffered saline-0.025%Triton for 30 min at room temperature, followed by overnight incubation with an anti-mouse alkaline phosphatase antibody (R&D systems, Minneapolis, MN, USA). Detection was done by Dylight 488 rabbit anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) with 1-h incubation at room temperature. Fluorescence imaging was performed using an inverted microscope (EVOS FL, Thermo Fisher Scientific). ALPL assay using p-nitrophenyl phosphate was performed as described before.

Scanning electron microscopy

For scanning electron microscopy (SEM), cleaned and sterilized disks in self-sealed pouches were received as such from the respective manufacturers. The disks were carefully mounted on stubs, sputter-coated, and viewed with Carl Zeiss AG-EVO® 40-series scanning electron microscope.

Statistical analysis

Statistical significance of the differences between the groups was determined using Student’s t test. The statistical power was calculated using the Biomath online software (http://www.biomath.info/power/index.html). We analyzed 10 samples for each group (alpha error 0.05), which resulted in a statistical power of 92%.

Blinding of the investigators

While performing the experiments, JM (first co-author) was not aware of the sources/manufacturers of the disks, which were identified by their size (small and large) only. At the end of the analyses, each disk’s manufacturer was revealed to her by JSD (first co-author).