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3.5.4 Apoptosis and celi cycle assay

Adherent MSCs were cultured in 60-mm dishes in a-MEM supplemented with 20% FBS until used for analysis. Cells were lifted by incubation in 0.05% trypsin-0.02% EDTA solution (Invitrogen), suspended in PBS, counted with a haemocytometer. The celi suspensions were washed in PBS and used for apoptosis assay with Annexin V:FITC Apoptosis Detection Kit I (BD Biosciences, Mississauga, Ontario, Canada) according to the manufactureris instructions or for celi cycle assay by incubation with staining buffer containing RNase 0.2 mg/mL (Roche Diagnostics, Laval, Quebec, Canada) and propidium iodide 25 pg/mL (BD Biosciences) in PBS for 45 min at 37°C. After incubation, fluorescence was measured for 10,000 events per sample by flow cytometry (BD AccuriCó, BD Biosciences) and analysis was performed using BD AccuriCó software (BD Biosciences) at day 7, 14, 21 of culture.

3.5.5 Proliferation assays

For proliferation assays, MC3T3-E1 cells were plated in 96-well plates (Sarstedt) at 2,500 cells/cm² and were cultured in a-MEM medium containing 10% FBS for 96 h. The adherent MSCs were left in 100-mm dishes to reach confluence, washed twice with PBS, and then harvested for experimentation. The MSCs were seeded at 20 000 cells/cm² in 96-well plates and were cultured in a-MEM medium containing 20% FBS for 96 h. For both cells types, medium was replaced for medium containing 2% FBS without or with 400, 500 and 600 ng/mL TSP-1 (R&D Systems, Minneapolis, MN, USA) or 10‘⁷, 10‘⁸, 10'⁹ M hexarelin (Abcam, Toronto, Ontario, Canada) for another 96h. Yiability was evaluated before treatment (day 0) as well 96 h after with tetrazolium microtiter assay (Sigma-Aldrich, Oakville, Ontario, Canada). Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to the medium at a finał concentration of 0.5 mg/mL. Two hours later, formazan crystals generated by cellular reduction of the MTT reagent were dissolved