Tenocyte Culture on Inflammatory Scaffolds

Hydrated, cross-linked scaffolds were soaked in growth media for 1 h at 37°C before being placed in ultra-low attachment 6-well plates (Fisher, Waltham, MA). Confluent tenocytes were trypsinized and resuspended in growth media at a concentration of 2.5 × 10⁵ tenocytes per 20 μL. Scaffold variants were seeded with tenocytes using a previously established static seeding method.^{35 (link)} The scaffolds were incubated for 15 min at 37°C following the addition of 10 μL of the cell suspension to one side of the scaffold. The scaffolds were then flipped over and another 10 μL of cell suspension was added for a total of 2.5 × 10⁵ tenocytes seeded on each scaffold. To allow for initial cell attachment, scaffolds were placed in the incubator for 2 h. After this period, additional media was added and scaffolds were incubated at 37°C and 5% CO₂ for 24 h before the media was changed for one of three inflammatory media variants. These variants included: (1) growth media (control), (2) media supplemented with 0.1 ng/mL of the pro-inflammatory factor interleukin-1 beta (IL-1β) (inflammatory), and (3) media supplemented with 1 ng/mL of IL-1β (high inflammatory). Scaffolds were cultured in culture media as described above, with or without the addition of serum, for the duration of the experiment. Media changes occurred every 3 days.