Heart valve replacement in the pediatric population is an unsolved problem. Although, in general, all heart valve replacements function well in adult and pediatric patients, there are several disadvantages, especially for children and young adults. The limitations of mechanical and biological heart valve prostheses include endocarditis, thromboembolic complications, early degeneration, and the lack of growth.1
The ideal heart valve replacement should not induce immunological reactions and should not be thrombogenic while providing ideal flow and biomechanical properties. It should integrate itself into the surrounding host tissue and have the potential to grow.2 Tissue engineering may have the potential to closely provide the characteristics mentioned above. First steps have been taken toward successful clinical application by the successful fabrication of tissue-engineered heart valves in vitro and the application in vivo.3–6
An optimal environment for tissue maturation is required to obtain vital, three-dimensional autologous tissue derived from cell-seeded heart valve scaffolds. In vivo conditions are simulated using a pulsatile bioreactor system that supplies cells with oxygen and cell culture medium containing growth factors. Meanwhile, cells are exposed to mechanical stress (e.g., pressure, strain, and compression) advocating extracellular matrix formation.7–9
Although the bioreactor represents a closed and sterile system, obtaining visual information about the opening and closing behavior of the tissue-engineered heart valve leaflets to administer optimal dynamic conditions and to provide reliable valve performance in vivo later is still important. In our previously described bioreactor,10 the opening and closing of the heart valve was visible, but the performance of the leaflets could not be accurately analyzed. We therefore developed a new combined monitoring and bioreactor device that allows optical monitoring and live imaging of the heart valve performance. We were able to analyze the exact movement of the leaflets during conditioning in a humidified incubator. With a camera module attached to the bioreactor, tissue-engineered heart valves could be observed during the entire in vitro phase and compared with native heart valve tissue. This setting helps find optimal dynamic conditions for tissue-engineered valves.
Materials and Methods
Bioreactor Design and Function
The combined system is made of acrylic glass and is completely transparent, allowing the observation of tissue development during in vitro conditioning. As shown in Figures 1 and 2, the optical device consists of an acrylic glass cylinder with a view window on top and a screw thread for camera objectives.
The bioreactor consists of three different chambers, the ventilation/air chamber (I) and the two cell culture medium chambers (II and III) as described previously.10 Chamber I is separated from chamber II by a silicone diaphragm and connected to an air-driven respirator pump (dual-phase control ventilator; Harvard Apparatus, Holliston MA). The cell culture medium continuously circulates through a closed-loop system.
This setting provides continuous pulsatile perfusion and mechanical stimulation of the tissue-engineered valve. By adjusting stroke volume, stroke rate, and inspiration/expiration time of the ventilator, various pulsatile flows and different levels of pressure can be obtained. The tissue-engineered valve is continuously perfused by cell culture medium that is circulating through the bioreactor. The bioreactor itself is connected to a reservoir (total volume 750 ml).
By pumping air into chamber I, the silicone diaphragm that separates chambers I and II is lifted up and pushes the cell culture medium through chambers II and III. Meanwhile, cell culture medium is pumped from chamber III into the reservoir. By sucking air out of chamber I, the silicone diaphragm becomes arched into chamber I, and cell culture medium from a reservoir flask is drawn into chamber II. We thus developed a bioreactor for optical monitoring of tissue-engineered valves with a closed-loop system for valve conditioning.
In our laboratory, we focus on using autologous cell sources (vascular cells, progenitor cells, etc.) and biodegradable polymeric scaffolds for heart valve fabrication.11 The polymer serves as a three-dimensional matrix until the cells produce their own extracellular matrix. The polymeric scaffold is seeded with autologous cells. For heart valve replacement, two types of cells are needed to closely imitate a native heart valve: myofibroblast-like cells producing extracellular matrix and endothelial-like cells serving as an antithrombogenic layer. Through biodegradation of the polymer and extracellular matrix formation, a new and completely autologous tissue can be generated.4,12,13
The bioreactor was placed in a standard humidified incubator at 37°C and 5% CO2 (HERAcell; W. C. Heraeus GmbH, Hanau, Germany) and connected to a reservoir flask filled with cell culture medium. The cell culture medium used was a 1:1 mixture of “Fibroblast Growth Medium” and “Endothelial Cell Growth Medium” (PromoCell GmbH, Heidelberg, Germany) with 10% “Fetal Calf Serum” (LonzaGroup Ltd, Basel, Switzerland). The connection between bioreactor, respirator, and reservoir was established by silicone tubes (Figure 4). Images of the opening and closing behavior of heart valves (Figure 3) were obtained using an onboard camera (Figure 2) (Olympus E420 with Olympus ZUIKO DIGITAL 35 mm f3.5 Macro objective, Olympus imaging Corp., Japan).
Bioreactor Design and Function
The flow rates ranged from 100 to 3000 ml/min, pressures ranged from 3 to 150 mm Hg, and shear stress ranged from 1.12 to 32.45 dyn/cm2. In our experiment, low systolic pressures (10–15 mm Hg) were used for optimal tissue conditioning. During conditioning, the in vitro performance of the tissue-engineered heart valve can be monitored with a digital camera and can be analyzed on a computer. Images of a native heart valve (porcine valve) and tissue-engineered heart valves could be compared (Figure 3). The system is sterile, isolated, and fits in a cell incubator; it can be sterilized using ethylene oxide.
All the constructs were tested for microbiological contamination after maturation in the bioreactor. In a series of 10 heart valves, we found one microbiological contamination. We previously described the successful fabrication of tissue-engineered heart valves in our bioreactor system.11 The biological characteristics of our tissue-engineered heart valves (e.g., morphology, immunohistochemistry, and electron microscopy) can be analyzed after explantation from the bioreactor. With our novel device, we were able to condition tissue-engineered heart valves and additionally test the hemodynamic performance of the heart valve constructs. The pressure gradient was measured in each heart valve. Hereby, a transvalvular gradient of <10 mm Hg (ΔPmax) was acceptable for our tissue- engineered heart valve construct.
There is a great demand for alternative heart valve replacements in cardiovascular surgery, especially for patients with congenital heart defects. The major disadvantage of currently used prostheses is that they have no capability for growth or repair. Therefore, pediatric patients commonly require a number of surgical procedures over the years.1,11 Tissue engineering potentially allows the creation of living autologous tissue for surgical purposes that is comparable with native tissue.14 In our laboratory, we are therefore developing tissue-engineered cardiovascular structures with the help of biodegradable polymeric scaffolds; these scaffolds represent a three-dimensional matrix for cell attachment and tissue growth.15,16 Autologous neotissue for implantation is created by a process in which cells are isolated from different cell sources, are seeded onto a polymeric scaffold, and subsequently form an extracellular matrix.11 For this approach, an optimal in vitro environment is required.7 It should not only provide sufficient supply of the construct with oxygen and cell culture medium but also biochemical and mechanical stimuli to favorably promote tissue maturation.17,18
Finding the right in vitro conditions is difficult.11 An optical control helps in calibrating the dynamic conditions and adapting them to the needs of the particular tissue-engineered construct. The bioreactor introduced here easily fits in a standard humidified incubator, minimizing the risk of contamination. The transparency allows visual control as well as live imaging of the tissue-engineered construct so that potential construct malfunction or contamination can be revealed.
All heart valve scaffolds are custom made and are not comparable with a commercially available off-the-shelf heart valve product.19 The scaffolds vary in thickness, porosity, and fabrication technique, resulting in different heart valve scaffolds and different leaflet performances.20
Our newly developed device is able to provide information on the optimal heart valve performance and the potential feasibility for implantation in an animal experiment. With the camera module attached to the bioreactor, tissue-engineered valves can be observed during the entire in vitro phase. This is an important step toward finding optimal dynamic conditions for tissue-engineered valves and to compare them with native heart valve tissue.
We developed a combined system for both heart valve maturation and optical monitoring to improve the in vitro conditioning for the tissue engineering of heart valves. However, there are still many aspects to be improved in the field of in vitro conditioning,21,22 e.g., the right choice of biopolymer, optimized cell distribution on the scaffold, and autologous cell culture media.
Supported by a grant from the German Ministry for Education and Research (BMBF 01GN0544).
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