Background and aim of the study: The generation of a functional, non-immunogenic, non-thrombogenic construct based on autologous cells seeded onto an acellular extracellular matrix is the major goal in heart valve tissue engineering. The study aim was to identify culturing conditions required to achieve a stable endothelial cell (EC) layer under physiological flow conditions, a prerequisite for the requested characteristics.
Methods: Eleven detergent-decellularized ovine pulmonary valves (PVs) were statically reseeded in special bioreactors with ovine venous ECs (1.2¥107 cells per valve). The dynamic culture was started with 0.1 l/min in eight bioreactors. In four bioreactors the initial flow rate was slow, and increased by 0.1 l/min twice each day until maximal flow was 0.5 l/min and pulsation rate (PR) was 20 beats/min; in four other bioreactors the flow was increased by 0.7 l/min/day and reached 2.0 l/min with a PR of 50 beats/min. The mean system pressure was maintained at 25 ± 5 mmHg during the whole dynamic cultivation in both groups. Three statically reseeded valves served as baseline. After achieving maximal appointed flow, the valves were investigated morphologically

(hematoxylin and eosin staining, electron microscopy, von Willebrand factor, endothelial nitric oxide synthase immunostaining) and for metabolic activity (MTS assay). Results: After reseeding, the endothelium appeared on the luminal surface of the PV as a non-confluent monolayer. Moderate pulsatile circulation induced complete confluence of EC monolayers on both cusp sides and the pulmonary wall. A high flow rate led to a partial loss of cells on the wall surface with large defects, and to complete cell wash-off from cusps. Cusp and wall metabolic activity was significantly higher after culture under moderate flow (p <0.001) than in other groups, and was absent from cusps in high-flow bioreactors.
Conclusion: Moderate pulsatile flow with small increments stimulates EC proliferation on the ovine decellularized valve scaffold. A rapid increase in bioreactor flow to physiological levels leads to significant damage of the reseeded endothelium and complete loss of cusp cellularity. This effect may be responsible for the in-vivo failure of static reseeded tissue-engineered valves exposed to physiological hemodynamic forces.
The Journal of Heart Valve Disease 2006;15:287-294