Mesencult basal media

BMSCs were generated from each experimental group of mice as previously described (Wu et al., 2006 (link)). In brief, BMSCs were separated by low-density gradient centrifugation from 3- to 4-week-old mice and then resuspended and cultured in mouse MesenCult medium (MesenCult basal media plus 20% of MesenCult Supplemental; Stem Cell Technologies) at 37°C and 5% CO₂. When the cultures reached 80%–90% confluence, cells were trypsinized and re-plated. BMSCs of identical passage number (between passages 3 and 5) were used for experiments.
For osteoblast differentiation, 5 × 10⁴ BMSCs were cultured for 7 days in 6-well plates using osteogenic differentiation medium (MesenCult medium supplemented with 10⁻⁷ M dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate). For induction of adipocyte differentiation in vitro, 1 × 10⁵ BMSCs were plated in 6-well tissue-culture plates and cultured with adipogenic differentiation medium (MesenCult medium supplemented with 10⁻⁷ M dexamethasone, 450  μM isobutylmethylxanthine, 1 μg/ml insulin, and 200 μM indomethacin). For chondrocyte differentiation assays, 1 × 10⁵ BMSCs were plated in 6-well plates and cultured with chondrogenic differentiation medium (osteogenic differentiation medium supplemented with 10 ng/ml transforming growth factor β3).

A composite bone material of approximate dimensions 3 mm × 4 mm × 3 mm was placed in a 10 cm² petri dish. To sterilize, the composite material was left in the open dish in the culture hood under UV light for 1 hour. BMSCs (StemCell Technologies, Vancouver, BC, Canada) were plated around the composite material at a density of 3 × 10⁵ cells/mL, and 3–4 mL of noncomplete MesenCult basal media (StemCell Technologies) was added. Cells were incubated at 37°C and 5% CO₂ for 3 days in a Forma Steri-Cycle CO₂ Incubator (Thermo Scientific) with images taken every 24 hours. At the end of 72 hours, the media and composite material were removed from the culture. Cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes and washed three times for 3 minutes each in PBS. ActinRed 555 and NucBlue Molecular Probes stains (Life Technologies, Carlsbad, CA, USA) were prepared in PBS as per company specifications and incubated on cells for 20 minutes. Cultures were then washed in PBS three times for 3 minutes and imaged using an Evos FL fluorescence microscope (Thermo Scientific).