Collagen gel contraction assay

Three-dimensional collagen gels were prepared by mixing 16 mL of chilled collagen solution (PureCol® Type I bovine collagen; Advanced BioMatrix, Sand Diego, CA), with 2 mL of sterile 10X PBS. While keeping the solution on ice, the pH was adjusted to 7.4 using sterile 0.1 M NaOH, and the volume was brought up to 20 mL with sterile water. Gels were cast in a 24-well plate by adding 600 µL solution to each well and allowing them to solidify at 37 °C, 5% CO₂ overnight. The following day, P0 fibroblasts cultured on 5 kPa elastic surfaces were passaged and seeded on the collagen matrices at a density of 1.5 × 10⁴ cells per well and allowed to adhere for 24 h. The gels were then released from the wells using a circular cutting tool and immediately infected with their corresponding adenoviral vector, or treated with 4 ng/mL recombinant human TGF-β₁ (Cell Signaling, #8915) as a positive control. Images were taken immediately following treatment, and subsequently every 24 h for a total of 72 h post-treatment. Gel contraction was estimated by measuring the surface area of the top of the gel using ImageJ image processing software [44].