Isolation and Characterization of Murine Periosteal Progenitors

Intact, age-matched mouse tibias were dissected free of surrounding tissues and both ends were removed at the growth plate sites. The remaining bone fragments were digested in 2 mg/mL collagenase A and 2.5 mg/mL trypsin. Cells from the first 3 minutes of digestion were discarded and cells from a subsequent 20 minutes of digestion were seeded in the growth medium (αMEM supplemented with 15% fetal bovine serum [FBS] plus 55 μM β-mercaptoethanol, 2mM glutamine, 100IU/ mL penicillin and 100 μg/mL streptomycin) for periosteal mesenchymal progenitor culture. For colony-forming unit fibroblast (CFU-F) assay, cells were seeded at 0.3 × 10⁶ cells/ T25 flask. Seven days later, flasks were stained with 3% crystal violet to quantify CFU-F numbers. To study the radiation effect on progenitor proliferation, cells were seeded at 5600cells/cm², irradiated (8Gy) the next day, and counted at 0, 1, 2, 4, and 5 days postradiation. To study the radiation effect on differentiation, cells were irradiated (8 Gy) when confluent, then switched to either osteogenic medium (αMEM with 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg/ mL ascorbic acid, 100IU/mL penicillin, and 100 μg/mL streptomycin) for 2 weeks followed by alizarin staining, or adipogenic medium (αMEM with 10% FBS, 0.5 mM isobutylmethylxanthine, 10 mM indomethacin, 1 μM dexamethasone, 10 μg/mL insulin, 100IU/mL penicillin, and 100 μg/mL streptomycin) for 1 week followed by Oil Red O staining. For chondrogenic differentiation, after radiation progenitor cells were suspended at 1.25 × 10⁶ cells/mL in chondrogenic medium (high-glucose DMEM, 100 μg/mL sodium pyruvate, 1% ITS+Premix, 50 μg/mL ascor-bate-2-phosphate, 40 μg/mL L-proline, 0.1 mM dexamethasone, 10 ng/mL TGF-β3,100 IU/mL penicillin, and 100 μg/mL streptomycin). Aliquots of 200 μL cell solution were then distributed to a V-bottomed 96-well plate followed by centrifugation at 300G for 5 minutes. Pellets were cultured for 3 weeks and then harvested for paraffin sections with Alcian blue staining. To culture cells under hypoxia condition, cell culture plates were placed in a Modular Incubator Chamber (Billups-Rothenberg, Del Mar, CA, USA) supplied with 0.1% oxygen.