2.21. Cell Scratch Assay (In Vitro Wound Healing)

The cell scratch assay was proposed for the assessment of the wound-healing properties of the 3D-printed patches, as a facile and low-cost in vitro method to evaluate the migration ability of cells [24]. Briefly, HDFa cells were seeded into 6-well plates, at an initial density of 2 × 10⁵ cells/well and incubated at 37 °C for 72 h (in high glucose DMEM with 10% v/v FBS). Two perpendicular scratches were generated in each well, via a sterile tip of a pipette (200 μL), as well as an intersection at a point close to the center of the well. The cross-shaped scratch was used as a location marker. After rinsing with PBS, the cells were incubated with three different concentrations of dissolved 3D-printed patches (0.1, 1 and 5 mg/mL 3DPect) and serum-free medium. The distribution and quantity of cells in the scratch area in each well were monitored under a microscope, and images were recorded at 0, 24 and 72 h, respectively. Wound width and wound closure were calculated according to Equations (4) and (5),

where, W_i and W_f are the initial and final wound width, respectively.

where, A_i and A_f are the initial and final cell-free area of the simulated wound, respectively.

Wound width was calculated as the average distance between the edges of the scratch. Manual quantification was carried out and the initial and final wound width was calculated as the average of 50 width measurements across the scratch. The wound area was calculated based on the cell-free area in captured images using the ImageJ public domain software (NIH, Bethesda, MD, USA).