A low-flow adaption phase improves shear-stress resistance of artificially seeded endothelial cells

Objectives: Evaluation of the effect of different adaption phases on the shear-stress resistance of artificially seeded endothelial cells (EC).

Material and Methods: Human EC, fibroblasts (FB), and smooth muscle cells (SMC) were obtained from vena saphena pieces. 15 poly-urethane grafts (24mm diameter) and 8 stentless porcine valves (Freestyle, Medtronic) were seeded with FB and SMC (44±12 million cells) followed by EC seeding (27±9 million cells). Shear-stress testing: perfusion assembly allowing pulsatile flow (80 pulses per min.). Adaption phase (AP): 0.9±0.3 l/min, systolic pressure: 40–50mmHg. Normal flow (NF): 3.2±0.6 l/min., systolic pressure: 140–160mmHg for four hours. The vascular grafts were divided into 3 groups (n=5 each): A (NF), B (AP: 15 minutes, then NF), and C (AP: 30 minutes, then NF). The porcine valves were divided into two groups (n=4 each): Group D (NF) and Group E (AP: 30 minutes, then NF). Specimens for immunohistochemical stainings (factor VIII, collagen IV, laminin, CD31, eNOS) and scanning electron microscopy were obtained after cell seeding, before and after perfusion.

Results: All grafts had a confluent EC layer before perfusion. Perfusion results: Groups A+D: large EC-layer defects, no FB and SMC on the buttom. Group B: few defects, confluent FB and SMC layer on the buttom. Groups C + E: very rare defects, confluent FB and SMC layer. Immunohistochemical stainings: viable EC (factor VIII/CD31(eNOS) and formation of a basal membrane (collagen IV/laminin).

Conclusions: An adaption phase of 30 minutes was sufficient to allow seeded EC to adapt to shear-stress. The basal membrane was important for the maintainance of a confluent EC layer under perfusion.