Osteogenic stimulatory supplement

Cells were cultured in six well plates containing 2 ml of MesenCult MSC Basal Medium (Human) (Stemcell technologies, cat. 05401) supplemented with Osteogenic Stimulatory Supplement (Stemcell technologies, cat. 05405), β-Glycerophosphate 1M (Stemcell technologies, cat. 05406), dexamethasone (Stemcell technologies, cat. 05407) and ascorbic acid (Stemcell technologies, cat. 07157) for 15 days (Osteogenic medium). The plates were kept in a humidified incubator at 37°C and 5% CO₂ and the culture medium was changed every three days. After the 15-day period, the medium was removed and the cells were washed with PBS. After fixation of the cells with PFA 4%, for 30 min the cells were washed three times with distilled water. Alizarin red S staining (Sigma, cat. A5533) was used to confirm osteogenic differentiation using manufacturer's protocol. Briefly, the cells were incubated in 2 ml solution of sodium alizarin at RT for 30 min. The dye was carefully removed and extensive washing with distilled water followed. The fixed and dyed cells were observed using optical microscope (Nikon Eclipse TE2000-U inverted microscope, Nikon Inc, Melville, NY, USA) and photographed [20] (link), [30] (link).

Pooled sera were added to the Mesenchimal Stem Cell Grow Medium (PromoCell, GMBH Heidelberg, Germany) at 10% of final concentration. Cells were plated at density of 5 × 10⁴ cells per well into 24-well plates. The osteogenic differentiation was performed with osteogenic medium containing Osteogenic Stimulatory Supplements (15% Stemcell), 10⁻⁸ M dexamethasone, 3.5 mM β-glycerophosphate, and 50 μg/mL ascorbic acid (StemCell Technologies Inc, Vancouver, British Columbia, Canada) Adipogenic differentiation was performed by using isobutylmethylxanthine (0.5 mM), indomethacin (200 μM), dexamethasone (10⁻⁶ M), and insulin (10 μg/mL) in basal medium. For both osteogenic and adipogenic differentiation, the medium was changed every 3 days after initial plating.

The osteogenic differentiation was obtained by using osteogenic medium containing 15% of Osteogenic Stimulatory Supplements (#05404, Stemcell), 10⁻⁸ M dexamethasone, 3.5 mM β-glycerophosphate, and 50 μg/mL ascorbic acid (StemCell Technologies Inc., Vancouver, BC, Canada), as previously reported [32 (link)]. The medium was changed every 3 days after initial plating. After 3, 7, and 14 days of osteogenic induction, cells were harvested and the pellet was processed or stored at −80 °C until use.

Sera, obtained from each runner before and after the competition, were mixed in two pools named PRE RUN and POST RUN sera, respectively. In order to study the induction of osteogenic and adipogenic differentiation, respectively, before and after physical exercise, we treated the BM-hMSC cell line (human bone marrow-human mesenchymal stem cells, PromoCell, Heidelberg, Germany) with pooled PRE RUN and POST RUN sera [5 (link)].
Pooled sera were added to the above cell line at 10% final concentration. Cells were plated at a density of 5 × 10⁴ cells per well into 24-well plates and cultured up to 21 days. In particular, osteogenic differentiation was performed with osteogenic medium containing osteogenic stimulatory supplements (15%, Stemcell Technologies Inc., Vancouver, Canada), 10⁻⁸ M dexamethasone, 3.5 mM β-glycerophosphate, and 50 μg/ml ascorbic acid (Stemcell Technologies Inc.). The adipogenic differentiation was performed by using 0.5 mM isobutylmethylxanthine, 200 μM indomethacin, 10⁻⁶ M dexamethasone, and 10 μg/ml insulin in basal medium. Chondrogenic differentiation was performed by culturing hMSCs with mesenchymal stem cell chondrogenic differentiation medium (PromoCell, Heidelberg, Germany). For osteogenic, adipogenic, or chondrogenic differentiation, the medium was changed every 3 days after initial plating.