Surgical marking pen dye inhibits saphenous vein cell proliferation and migration in saphenous vein graft tissue

Abstract

Objective: Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Because little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration and proliferation, which are responses to injury after grafting.

Methods: Fresh human saphenous veins were obtained as residual specimens from leg bypass surgeries. Portions of the vein that had been surgically marked with crystal violet were analyzed separately from those that had no dye marking. In the laboratory, they were split into easily dissected inner and outer layers after removal of endothelium. This cleavage plane was within the circular muscle layer of the media. Cell migration from explants was measured daily as either (1) percentage of migration-positive explants, which exclusively measures migration, or (2) number of cells on the plastic surrounding each explant, which measures migration plus proliferation. Cell proliferation and apoptosis (Ki67 and TUNEL staining, respectively) were determined in dye-marked and unmarked areas of cultured vein rings. The dose-dependent effects of crystal violet were measured for cell migration from explants as well as for proliferation, migration, and death of cultured outer layer cells. Dye was extracted from explants with ethanol and quantified by spectrophotometry.

Results: There was significantly less cell migration from visibly blue compared with unstained outer layer explants by both methods. There was no significant difference in migration from inner layer explants adjacent to blue-stained or unstained sections of vein because dye did not penetrate to the inner layer. Ki67 staining of vein in organ culture, which is a measure of proliferation, progressively increased up to 6 days in nonblue outer layer and was abolished in the blue outer layer. Evidence of apoptosis (TUNEL staining) was present throughout the wall and not different in blue-stained and unstained vein wall segments. Blue outer layer explants had 65.9 ± 8.0 ng dye/explant compared with 2.1 ± 1.3 for nonblue outer layer explants. Dye applied in vitro to either outer or inner layer explants dose dependently inhibited migration (IC50∼10 ng/explant). The IC50s of crystal violet for outer layer cell proliferation and migration were 0.1 and 1.2 μg/mL, whereas the EC50 for death was between 1 and 10 μg/mL.

Conclusions: Crystal violet inhibits venous cell migration and proliferation, indicating that alternative methods should be considered for marking vein grafts.