Primary hMSCs (Lonza Inc., Allendale, NJ) were expanded in proliferation medium composed of Dulbecco’s modified Eagle’s medium (DMEM; Corning Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 2 mM l-glutamine (Life Technologies, Carlsbad, CA), and penicillin (100 IU/ml)/streptomycin (100 μg/ml; Life Technologies).

2D culture. hMSCs of passage 3 to 5 were plated at 5000 cells per well in 12-well plates, grown until 80 to 90% confluent, and then transduced with and without an AdOPG and RFP in DMEM at a MOI of 200 and polybrene (4 μg/ml; Sigma-Aldrich, St. Louis, MO). Twenty-four hours after transduction, hMSCs were subjected to differentiation medium consisting of proliferation medium plus 10 mM β-glycerophosphate, ascorbic acid (50 μg/ml), and 0.1 μM dexamethasone. Cell cultures were evaluated on day 7 after transduction for morphological changes, transduction efficiency, and Western blot.

Osteogenic differentiation of hMSCs on Col-GAG and MC-GAG. hMSCs (3 × 105) were seeded onto 8-mm discs of CG-GAG and MC-GAG scaffolds in proliferation medium. The cells were resuspended in growth medium, and half of the suspension was used to seed one side of the scaffold. After incubation for 15 min, the scaffold was turned upside down and the other half of the suspension was used to seed the opposite side. One millimeter of growth medium was then added to each well. Twenty-four hours after seeding, the medium was switched to osteogenic differentiation medium consisting of 10 mM β-glycerophosphate, ascorbic acid (50 μg/ml), and 0.1 μM dexamethasone.

Indirect hMSC and hOC cocultures. hMSCs (2 × 105) were seeded to 6 mm of Col-GAG and MC-GAG scaffolds in proliferation medium. Twenty-four hours after seeding hMSCs, primary hOC precursors (6 × 104; Lonza Inc., Allendale, NJ) were cultured in Osteoclast Precursor Basal Medium (Lonza, Allendale NJ) supplemented with M-CSF (33 ng/ml), RANKL (66 ng/ml), 10 mM β-glycerophosphate, ascorbic acid (50 μg/ml), and 0.1 μM dexamethasone for concurrent hMSC and hOC differentiation on 24-well Corning Osteo Assay Surface Microplates (Corning, NY). After 2 hours, Col-GAG and MC-GAG scaffolds were transferred to 8-μm Transwell inserts (Corning, NY) and cocultured with hOCs. Medium was changed every 3 days for 3 weeks.

Direct hMSC and hOC cocultures. hMSCs (3.5 × 105) were seeded to 8 mm of Col-GAG and MC-GAG scaffolds in proliferation medium. Twenty-four hours after seeding hMSCs, hOCs (6 × 104) were cultured in Osteoclast Precursor Basal Medium (Lonza Inc., Allendale NJ) supplemented with M-CSF (33 ng/ml), RANKL (66 ng/ml), 10 mM β-glycerophosphate, ascorbic acid (50 μg/ml), and 0.1 μM dexamethasone on 24-well Osteo Assay Microplates. After 2 hours, Col-GAG and MC-GAG scaffolds were transferred to the Osteo Assay Microplates and directly cocultured with hOCs. Medium was changed every 3 days for 2 weeks.

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