2.4. MC3T3-E1 cell culture and encapsulation

A murine pre-osteoblast cell line MC3T3-E1 (ATCC, CRL-2593) was expanded in alpha-MEM media (Gibco) with 10% fetal bovine serum (FBS, Atlanta Biologicals) and 50 U/mL penicillin, 50 μg/mL streptomycin (1% P/S, Corning). MC3T3-E1 cells were cultured to ~90% confluency and then collected using 0.05% trypsin/EDTA (Gibco). Cells were combined at 2×10⁷ cells per mL precursor solution and polymerized as described above. Cell-laden hydrogels were cultured in 24-well plates as mono-cultures or in a 24-well transwell inserts with RAW 264.7 or primary murine macrophages (see below) seeded at the bottom of the well plates as co-cultures. The mono-culture and co-cultures were placed in osteogenic differentiation media containing alpha-MEM, 10% FBS, 1% P/S, supplemented with 10 mM β-glycerophosphate (Sigma), 0.1 mM dexamethasone (Sigma), and 50 μg/ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma). The medium was supplemented or not with 1 μg/mL lipopolysaccharide from E. coli (LPS-EB O111:B4, standard purity, Invitrogen).