Tissue engineering of pulmonary heart valves.

Citation:

Steinhoff G, Stock U, Karim N, Mertsching H, Timke A, Meliss RR, Pethig K, Haverich A, Bader A. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits in vivo restoration of valve tissue. Circulation. 2000;102:III-50-III-55.

This German group has developed an alternative to biodegradable polymer scaffolds for restoring heart valve tissue. They sought to address the issues of inflammation, thrombogenicity, and long-term durability associated with these scaffolds and developed a method for utilizing biological valves for regenerating heart valve tissue. By seeding acellularized heart valve matrices with autologous sheep cells, the researchers hypothesized that they could reconsitute heart valve tissue in vivo after in vitro processing. Pulmonary valves were removed from lambs and treated with trypsin and EDTA to acellularize them. Autologous cell culture of both endothelial cells and myofibroblasts began by obtaining the right carotid artery of one month-old lambs. By filling the artery with collagenase and subsequently flushing it with medium, endothelial cells could be obtained by centrifuging and resuspending the pellet. The endothelial cells were then cultured and kept at 37 °C. Myofibroblasts grew in culture dishes containing the remaining bits of arterial wall and were subsequently cultured. To reconstitute the cell surface of the valve walls, the valves were initially seeded with myofibroblasts, and then seeded with endothelial cells. Pulmonary valve replacement was performed on two groups of lambs. The first group received tissue-engineered right carotid arteries, while the control group received allogenic acellularized valves that had not been reseeded after acellularization. All animals survived the procedures, and only one control animal died later because of thrombosis. Echocardiography was utilized to monitor the animals periodically in the twelve weeks following transplant. Investigators examined the biological performance of the valves by measuring valve diameters and regurgitation. The valves were graded on degree of thickening of the wall and functionality. Tissue-engineered animals had no pulmonary regurgitation but a few had thickening of the walls without losing functionality. The control group had normal valve morphology but exhibited varying degrees of pulmonary regurgitation. After termination the valves were removed and examined using macroscopic dissection, histology and immunohistology methods. Macroscopic examination of the excised valves showed overall normal valve morphology. No valvar calcification was present in the seeded animals while all animals showed some subvalvar calcification and no supravalvar calcification. For histology the samples were fixed, dehydrated, embedded, sectioned, and stained. The sections were examined using light microscopy. For immunohistochemistry, the samples were frozen, sectioned, and treated with antibodies specific for endothelial cells
(vWF) and myofibroblasts (α-actin). The acellularization process was successful, and in vitro seeding resulted in a patchy surface of endothelial cells and myofibroblasts (positive vWF and α-actin). The unseeded controls showed an almost completely reconstituted endothelial layer (positive vWF) but virtually no myofibroblast growth. The tissue-engineered valves were completely cellularized with an equal distribution of endothelial cells and myofibroblasts by weeks 4 and 12. Furthermore, staining for procollagen I indicated that matrix was being synthesized. There was some inflammation at the site but it was greatly diminished by the twelfth week. By developing this revolutionary method, the investigators were able to overcome many of the difficulties associated with cell adhesion and tissue reorganization in synthetic grafts. This particular in vitro acellularization method maintains the proteins of the extracellular matrix that other studies have proven are necessary for enhanced cell adhesion. The in vivo results demonstrate that the heart valve tissue can be reconstituted three-dimensionally and that the endothelial layer and myofibroblasts will form after implantation. However, significant questions remain after this initial investigation. Most importantly, the long-term viability of these grafts remains unknown. The thickening of the walls seen in the initial three months after implantation did not affect valve function, in the tissue-engineered grafts but over time could lead to valve failure. Furthermore, the success of utilizing other cell types in this method is unknown and must be tested. Also, the possibility of redifferentiation of seeded cells, as affected by a microenvironment which may not be identical to normal conditions, may result in tissue populated with non-proliferating cells. Though short-term matrix reconstitution was shown by the presence of procollagen, the long-term ability to reconstitute the ECM with all growth factors and other necessary components is unknown. Tissue reorganization is another prerequisite for viable graft application, and it was shown that the in vitro seeding of the valve before implantation is necessary for tissue and matrix regeneration. It should also be noted that in vitro seeding is also a factor in preventing inflammation and calcification. Though this initial study shows significant success in the in vivo implantation of in vitro-seeded grafts, the issues of mechanical stability, long-term viability, seeding technique, and xenogenic valves must still be addressed. Cardiovascular disease currently affects over 22% of the American population. There has also been an alarming increase in the occurrence of obesity and type II diabetes, which are accompanied by atherosclerotic vascular disease and hypertension, among other complications. As the risk factors for these disease increase in even younger generations, cardiovascular disease continues to rise and viable long-term treatments are needed even more. This article develops a revolutionary new method for preparing vascular grafts that move beyond many of the issues seen with synthetic polymer grafts. If further studies and long-term trials can be performed, they may prove to be the most successful option for treatment of cardiovascular disease.