Srp3276

Biodegradable TEVGs (4 mm diameter, ≈400 μm thickness and 4 cm length) were fabricated using emulsion electrospinning (Nanon-01A, MECC, Tokyo, Japan) from PHBV (403105, Sigma-Aldrich, Saint Louis, MO, USA): PCL (440744, Sigma-Aldrich, Saint Louis, MO, USA) (5:10%)/chloroform (366927, Sigma-Aldrich, Saint Louis, MO, USA) solution using the following parameters: 23 kV voltage, 0.5 mL/h feed rate, 2 mm rotating drum diameter, 22G needle, and 150 mm tip-to-collector distance. Abovementioned polymer ratio was determined in our previous studies [25 (link),31 (link),32 (link)]. In all these investigations, PHBV/PCL vascular grafts did not show any signs of dissolution as long as 1 year after implantation into rat abdominal aorta. Either VEGF (V7259, Sigma-Aldrich, St. Louis, MO, USA), bFGF (SRP4037, Sigma-Aldrich, St. Louis, MO, USA), or SDF-1α (SRP3276, Sigma-Aldrich, St. Louis, MO, USA) were dissolved in phosphate buffered saline (10010023, Thermo Fisher Scientific, Waltman, MA, USA) to 10 µg/mL concentration and then added to PHBV/PCL/chloroform solution (1:20), with the final concentration of 500 ng/mL. Grafts with the combination of VEGF, bFGF, and SDF-1α were two-layered, with the inner layer fabricated using 27G needle and containing VEGF (500 ng/mL) and the outer layer prepared utilizing 22G needle and containing bFGF and SDF-1α (500 ng/mL each).

Chemotaxis assay was performed following a standard procedure^{61 (link)} using heparin acrylic beads (Sigma, H5263) coated with 1 μg/mL purified human stromal cell-derived factor-1 (Sigma, SRP3276). Cell motility and chemotaxis were analyzed using ImageJ (https://imagej.nih.gov) analysis tools, as described previously^{53 (link),60 (link)}. Briefly, each individual cell was manually tracked using the Manual Tracking plugin of ImageJ, the data were collected and analyzed using the Chemotaxis plugin of ImageJ. Cell morphology was assessed by deploying the circularity index (complete circle = 1) and estimated by ImageJ analysis tools. Cell dispersion was analyzed using Delaunay triangulation algorithm (ImageJ Plugins) and was plotted as average explant triangle area, as described before^{54 (link)}. Cell protrusive area was analyzed in neural crest cells at the edge of an explant measuring the outgrowing area that derives from two consecutive timeframes with 4 min time interval; these two consecutive frames were subtracted to generate the new area^{12 (link)}. For analysis of cell chemotaxis and cell dispersion 10–15 explants were analyzed per condition for each independent experiment. For cell motility, cell morphology and cell protrusions 15–25 NCCs were analyzed per condition per experiment.