Inflammatory Challenge of hMSC-Seeded Scaffolds

Hydrated, cross-linked scaffolds were soaked in growth media overnight at 37 °C before being tapped dry on a Kimwipe and placed in ultralow attachment 6-well plates (Fisher, Waltham, MA). Confluent hMSCs were trypsinized and resuspended in growth media at a concentration of approximately 78 000 cells per 20 μL in preparation for static seeding.^{36 (link)} Ten microliters of the cell suspension were added to one side of the scaffolds. Then the scaffolds were incubated for 15 min at 37 °C and flipped, and another 10 μL of cell suspension was added. To allow for initial cell attachment, scaffolds were placed in the incubator for 2 h before additional growth media was added. Scaffolds were incubated at 37 °C and 5% CO₂ for 24 h before the media was exchanged for one of three inflammatory challenge media formulations that was then replaced every 3 days throughout the experiment: (1) control, growth media; (2) inflammatory, media supplemented with 0.1 ng/mL of the pro-inflammatory factor interleukin-1 β (IL-1β) and 1 ng/mL tumor necrosis factor α (TNFα); (3) highly inflammatory, media supplemented with 1 ng/ mL of IL-1β and 10 ng/mL TNFα.