Bone Marrow Stromal Cell Isolation and Osteoblast Differentiation

Bone marrow was flushed from femurs and tibias of mice at specified ages and pooled between like groups. Cells were immediately seeded into six-well plates at 1 × 10⁷ cells per well, 12-well plates on type 1 collagen–coated glass coverslips at 4 × 10⁶ cells per well, or 96-well plates at 5 × 10⁵ cells per well in basal culture medium (a-MEM, 20% FBS, 1% antibiotic/ antimycotic [Invitrogen, Carlsbad, CA, USA; #15240–062], 1% nonessential amino acids), osteogenic culture medium (basal culture medium + 50 µg/mL ascorbic acid, 10 mM beta glycerol phosphate, ±10⁻⁷ M dexamethasone [Dex], as indicated), or adipogenic culture medium (basal culture medium +1 µM rosiglitazone, Sigma-Aldrich). Media were changed every 3 days after seeding, and BMSC were selected by their ability to adhere to the plate after the first 3 days in culture. BMSC were cultured for up to 21 days in osteogenic medium to promote osteoblastic differentiation. In a subset of experiments, cells were exposed to 5 µM carbenoxolone during osteogenic culture, as described.^{(33 (link),34 (link))} Demarrowed cortical bone diaphyses were digested with collagenase as described to isolate cortical bone osteoblasts.^{(35 (link))} Cells were grown for 6 days in culture medium (a-MEM, 5% FBS, 5% calf serum [Hyclone Laboratories, Logan, UT, USA], 1% penicillin/streptomycin [Gibco, Grand Island, NY, USA]) before protein extracts were made.