2.6.2. Seeding onto NFMs

Electrospun PCL NFMs were sterilized by UV-O treatment. All the surface functionalization and TNF-α antibody immobilization steps were performed in sterile conditions. For the biofunctionalized NFMs, antibody immobilization was performed overnight, and after the BSA

#### *Nanomaterials* **2019**, *9*, 567

blocking step, cell seeding was performed. Activated and biofunctionalized electrospun NFMs were used as controls.

Confluent hACs were detached from the culture flask using trypsin, counted on a hemocytometer and seeded at a density of 200.000 cells/NFM. Seeding was performed using the droplet method, using a 50 μL drop of cell suspension on all sample groups in 24-well plates. The plates were placed at 37 ◦C and 5% CO2 over 4 hours. After cell attachment to the NFMs, 1 mL of expansion medium was added to each well. Afterwards, the medium was changed into differentiation medium. The seeding was performed in three independent experiments. For each independent experiment, constructs were cultured for 1, 14, 21 and 28 days under static conditions and collected at each time point for quantification of cell viability, DNA, total protein analysis, as well as GAGs deposition. Cell morphology was evaluated by SEM.
