Tissue engineering of heart valves: biomechanical and morphological properties of decellularized heart valves.

BACKGROUND AND AIM OF THE STUDY: Biological scaffolds are widely used in the process of cardiac valve tissue engineering. Scaffold characteristics are decisive for valve durability. Herein, the influence of three different decellularization protocols on the morphological and biomechanical properties of porcine pulmonary valve conduits was evaluated. METHODS: Pulmonary valve conduits were decellularized with 1% sodium deoxycholate (SD), 1% sodium dodecylsulfate (SDS), or 0.05% trypsin/0.02% EDTA. The degree of decellularization and morphological integrity of the treated pulmonary valve cusp, wall and myocardial cuff were analyzed with hematoxylin and eosin staining, Movat-Pentachrome staining, electron microscopy, and DNA assay. The conservation of extracellular matrix (ECM) proteins was evaluated by immunohistochemical staining against collagens I and IV, and laminin. The biomechanical properties of the obtained scaffolds were evaluated using uniaxial tension tests. Native grafts served as controls. RESULTS: All treatments resulted in complete decellularization of the cusp, whereas only SD and SDS treatments were able to remove completely all cells from the pulmonary valve wall and subvalvular myocardial cuff. The morphological integrity and preservation of ECM proteins was clearly superior in both detergent-treated groups. Enzyme treatment resulted in destruction of the basement membrane. Wall longitudinal tension parameters (stiffness, elasticity modulus, ultimate force; stress and strain) were significantly inferior in the trypsin/EDTA group (p < 0.05). No significant differences were observed between detergent-treated and native samples. The results of transversal tension parameters were comparable in all groups. CONCLUSION: Both, SD and SDS treatment of the pulmonary valve may better preserve the morphological and biomechanical properties of the scaffold than the chosen enzymatic treatment. In the authors' opinion, detergent-based decellularization should be used in preference to enzyme treatment in the tissue engineering of heart valves.

In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions.

The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.

Flow-dependent re-endothelialization of tissue-engineered heart valves.

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 (1.2x10(7) 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.