Background-Tissue engineering using in vitro-cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. Methods and Results-Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells.After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface.The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (␣-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. Conclusions-The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.

The shortage of human donor valves and disadvantages of current mechanical and xenogenic valve grafts cause a growing demand for biologically engineered valves. We developed a bioreactor for in vitro transformation of porcine semilunar heart valves into human valves. The reactor design was optimized in order to achieve a complete removal of porcine cells by trypsinization and cellularization of the remaining matrix with human vascular cells. The physical parameters of the reactor were characterized. Based on these data, decellularization and cellularization protocols were developed and the successful application of protocols was investigated by immunohistochemistry. The resulting reactor consisted of two connectable and perfuseable modules. Heart valve structures of varying sizes could be mounted into the reactor and could be completely depopulated within 4 days by a sequence of enzymatic and mechanical treatments applied simultaneously to the valves. Complete cellularization with human cells could be achieved after optimizing the seeding design, density, and time.

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Background —Tissue engineering using in vitro–cultivated autologous vascular wall cells is a new approach to biological heart valve replacement. In the present study, we analyzed a new concept to process allogenic acellular matrix scaffolds of pulmonary heart valves after in vitro seeding with the use of autologous cells in a sheep model. Methods and Results —Allogenic heart valve conduits were acellularized by a 48-hour trypsin/EDTA incubation to extract endothelial cells and myofibroblasts. The acellularization procedure resulted in an almost complete removal of cells. After that procedure, a static reseeding of the upper surface of the valve was performed sequentially with autologous myofibroblasts for 6 days and endothelial cells for 2 days, resulting in a patchy cellular restitution on the valve surface. The in vivo function was tested in a sheep model of orthotopic pulmonary valve conduit transplantation. Three of 4 unseeded control valves and 5 of 6 tissue-engineered valves showed normal function up to 3 months. Unseeded allogenic acellular control valves showed partial degeneration (2 of 4 valves) and no interstitial valve tissue reconstitution. Tissue-engineered valves showed complete histological restitution of valve tissue and confluent endothelial surface coverage in all cases. Immunohistological analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (α-actin), and matrix synthesis (procollagen I). There were histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, but these were not found in valve and pulmonary artery tissue. Conclusions —The in vitro tissue-engineering approach using acellular matrix conduits leads to the in vivo reconstitution of viable heart valve tissue.

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