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.
