Studies On Tissue Engineering Heart Valve Construction With Human Endothelial Progenitor Cells Transfected By Ad.FnCBD₆₄

Objective: To promote adhesion of endothelial progenitor cells(EPCs) to decellularized porcine aortic valve scaffle using gene transfection technique and to optimize TEHV construction in pulsatile bioreactor. Methods: Isolated and cultured human EPCs from bone marrow sample with Ficoll gradient centrifugation and differential adhesion culture in sequence, meanwhile, built a new pulsatile bioreactor with T-PLS pump system and a modified air-liquid exchange passage. Then constructed a replication-deficient recombinant adenovirus coding Fn signal peptide and FnCBD₆₄, and verified that the virus could efficiently express and secret FnCBD₆₄ after cell transfection using Western-blot and ELISA technique. After that, evaluated the effect of EPCs adhesion after Ad.FnCBD64 transfection, and did some primary study on TEHV culture in the newly built bioreactor. Results: EPCs is highly proliferative and it differentiates into endothelial cells in vitro. The bioreactor built could generated 0-6000ml/min pulsatile flow and could support TEHV culture in vitro. When transfected by Ad.FnCBD₆₄, EPCs efficiently express and secrete FnCBD₆₄, and that result in higher adhesion speed and stronger adhesion strength in transfected EPCs than the control group cells(P<0.05). When exposed to pulsatile flow in bioreactor, more Ad.FnCBD₆₄ transfected EPCs remained in situ in low-flow(0-600ml/min) hydrodynamics and almost no cell remained in high-flow (0-4800ml/min) environment. Conclusions: EPCs is the better choice of seed cell for TEHV construction and Ad.FnCBD₆₄ transfection efficiently promote adhesion of EPCs on decellularized xenograft aortic valves. Still, much work is needed in the future study of TEHV.