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In-vitro Evaluation einer neu entwickelten bio-hybriden tissue-engineerten Aortenklappenprothese zur TAVI-Applikation
In-vitro Evaluation einer neu entwickelten bio-hybriden tissue-engineerten Aortenklappenprothese zur TAVI-Applikation
During the last few years, transcatheter aortic valve implantation (TAVI) has been established as the initial therapy for patients with aortic valve diseases. The prostheses however, are limited in functionality and durability due to their production and implantation process. In this study, newly developed, tissue engineered aortic valve prostheses were analyzed and evaluated. The functionality and integrity of the decellularized and stented heart valves were evaluated after conditioning and in vitro TAVI simulation. The biohybrid aortic valves (BAV; n=6) were composed of decellularized homograft cusps and polyurethane patches sewed into Co-Cr-stents. The BAVs were colonized and cultivated with fibroblasts (FB) and subsequently with endothelial cells (EC), which were extracted from saphenous vein segments. After cell seeding, the valves were conditioned in a pulsatile bioreactor (500ml/min, 5days). Simulation of the TAVI intervention was performed by crimping and subsequent redilatation of the BAVs. The functionality of the BAVs was tested at increased flow conditions (1100ml/min, 2days). The valve function was visually monitored during this process using video imaging. Samples of the BAVs were taken before and after each step and analyzed using scanning electron microscopy (SEM), immunocytochemistry (ICC), immunohistochemistry (IHC) and Live/Dead® staining methods. After cell seeding, static cultivation and first conditioning confluent cell layers were observed in SEM. Positive IHC-staining with CD-31 (EC-specific antibody) and TE-7 (FB-specific antibody) indicated the presence of EC and FB on the surfaces of the BAVs. The construction of extracellular matrix (ECM) was verified through IHC-staining against Collagen IV and Fibronectin. A strong establishment of ECM was detected, especially after the first conditioning phase. However, a large number of lethal cells were detected by Live/Dead®-staining after crimping. Extensive regions of damaged cell-layers were detected by SEM-analysis. Furthermore, highly expressed ICAM staining was seen after perfusion. During conditioning and perfusion, the proper opening and closing behavior of the BAVs were visually monitored. The biohybrid aortic valve proves as an effective scaffold for tissue engineering. However, it is suggested to reconsider the crimping of tissue engineered aortic valves, since the procedure leads to obvious and severe damage to the tissue engineered cell layers.
Tissue Engineering, TAVI, Aortenklappe
Lee, Jang-Sun
2018
German
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Lee, Jang-Sun (2018): In-vitro Evaluation einer neu entwickelten bio-hybriden tissue-engineerten Aortenklappenprothese zur TAVI-Applikation. Dissertation, LMU München: Faculty of Medicine
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Abstract

During the last few years, transcatheter aortic valve implantation (TAVI) has been established as the initial therapy for patients with aortic valve diseases. The prostheses however, are limited in functionality and durability due to their production and implantation process. In this study, newly developed, tissue engineered aortic valve prostheses were analyzed and evaluated. The functionality and integrity of the decellularized and stented heart valves were evaluated after conditioning and in vitro TAVI simulation. The biohybrid aortic valves (BAV; n=6) were composed of decellularized homograft cusps and polyurethane patches sewed into Co-Cr-stents. The BAVs were colonized and cultivated with fibroblasts (FB) and subsequently with endothelial cells (EC), which were extracted from saphenous vein segments. After cell seeding, the valves were conditioned in a pulsatile bioreactor (500ml/min, 5days). Simulation of the TAVI intervention was performed by crimping and subsequent redilatation of the BAVs. The functionality of the BAVs was tested at increased flow conditions (1100ml/min, 2days). The valve function was visually monitored during this process using video imaging. Samples of the BAVs were taken before and after each step and analyzed using scanning electron microscopy (SEM), immunocytochemistry (ICC), immunohistochemistry (IHC) and Live/Dead® staining methods. After cell seeding, static cultivation and first conditioning confluent cell layers were observed in SEM. Positive IHC-staining with CD-31 (EC-specific antibody) and TE-7 (FB-specific antibody) indicated the presence of EC and FB on the surfaces of the BAVs. The construction of extracellular matrix (ECM) was verified through IHC-staining against Collagen IV and Fibronectin. A strong establishment of ECM was detected, especially after the first conditioning phase. However, a large number of lethal cells were detected by Live/Dead®-staining after crimping. Extensive regions of damaged cell-layers were detected by SEM-analysis. Furthermore, highly expressed ICAM staining was seen after perfusion. During conditioning and perfusion, the proper opening and closing behavior of the BAVs were visually monitored. The biohybrid aortic valve proves as an effective scaffold for tissue engineering. However, it is suggested to reconsider the crimping of tissue engineered aortic valves, since the procedure leads to obvious and severe damage to the tissue engineered cell layers.