Dual cell seeding and the use of zymogen tissue plasminogen activator to improve cell retention on polytetrafluoroethylene grafts

Objective. The purpose of this study was to enhance the retention of seeded endothelial cells (EC) on prosthetic vascular grafts. Dual-layer EC and sm ooth muscle cell (SMC) seeding and gene transfer of a zymogen tissue plasmi nogen activator gene (tPA) into seeded EC were studied. Methods. Polytetrafluoroethylene (PTFE) grafts were precoated with fibronec tin, seeded with SMC followed by EC a day later, and then, 24 hours later, exposed to an in vitro flow system for 1 hour. Cell retention rates were de termined for grafts seeded with EC only, a dual layer of EC on top of SMC, EC transduced with wild-type tPA, and EC transduced with zymogen tPA. Results. Seeding efficiency of PTFE pretreated with fibronectin was 260 +/- 8 cell/mm(2). After exposure to flow, only 39% +/- 14% of the EC were reta ined when EC were seeded alone, whereas 73% +/- 22% of EC remained on graft s when EC were seeded on top of SMC (P <.001, n = 10). The enzyme activity of a mutant zymogen tPA in absence of fibrin was 14 +/- 1 IU/mL, which is 3 .6-fold lower than that in the presence of fibrin (50 +/- 19 IU/mL), wherea s fibrin has no effect on the wild-type tPA activity. EC expressing a high level of wild-type tPA had a lower retention rate (37%) when compared with normal EC (45%). EC expressing the mutant zymogen tPA had an improved reten tion rate (54%, P =.001, n = 10) in absence of fibrin, whereas its retentio n rate was reduced to 43% when the cells were exposed to fibrin. Conclusion: SMC seeded between EC and PTFE improves EC retention in vitro. Transduction of zymogen tPA increases thrombolytic ability of seeded cells with less adverse impact on cell retention than wild-type tPA.