RESUMEN
BACKGROUND/OBJECTIVE: Ischemic heart disease is a major cause of mortality worldwide. Myocardial tissue engineering aims to create transplantable units of myocardium for the treatment of myocardial necrosis caused by ischemic heart disease - bioreactors are used to condition these bioartificial tissues before application. METHODS: Our group developed a multimodal bioreactor consisting of a linear drive motor for pulsatile flow generation (500 ml/min) and an external pacemaker for electrical stimulation (10 mA, 3 V at 60 Hz) using LinMot-Talk Software to synchronize these modes of stimulation. Polyurethane scaffolds were seeded with 0.750 × 106 mesenchymal stem cells from umbilical cord tissue per cm2 and stimulated in our system for 72 h, then evaluated. RESULTS: After conditioning histology showed that the patches consisted of a cell multilayer surviving stimulation without major damage by the multimodal stimulation, scanning electron microscopy showed a confluent cell layer with no cell-cell interspaces visible. No cell viability issues could be identified via Syto9-Propidium Iodide staining. CONCLUSIONS: This bioreactor allows mechanical stimulation via pulsatile flow and electrical stimulation through a pacemaker. Our stem cell-polyurethane constructs displayed survival after conditioning. This system shows feasibility in preliminary tests.
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Células Madre Mesenquimatosas/citología , Miocitos Cardíacos/citología , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Reactores Biológicos , Supervivencia Celular , Células Cultivadas , Estimulación Eléctrica/instrumentación , Diseño de Equipo , Humanos , Miocardio/citología , Poliuretanos/química , Flujo PulsátilRESUMEN
INTRODUCTION:: Decellularization of thick tissue is challenging and varying. Therefore, we tried to establish a multifactorial approach for reliable aortic wall decellularization. METHODS:: Porcine aortic walls were decellularized according to different procedures. Decellularization was performed for 24 (G1), 48 (G2), and 72 h (G3) with a solution of 0.5% desoxycholate and 0.5% dodecyl sulfate. The procedure was characterized using intermittent washing steps, the inclusion of sonication as well as DNase and α-galactosidase treatment. The decellularization efficiency was measured by the evaluation of 4',6-diamidino-2-phenylindole and hematoxylin and eosin staining and quantitative DNA assays. Pentachrome and picrosirius red staining, scanning electron microscopy as well as glycosaminoglycan assays were performed to evaluate the effect of the procedure on the extracellular matrix. RESULTS:: 4',6-Diamidino-2-phenylindole and hematoxylin and eosin staining revealed a large amount of remaining nuclei in all groups. However, consecutive DNase treatment had a significant effect. While the remaining DNA was detected in some samples of G1 and G2, samples of G3 were fully decellularized. Glycosaminoglycan content was significantly reduced to 50% after 24 h (G1) but remained constant for G2 and G3. Picrosirius red staining revealed an intact and stable collagen network without any visible defects. Pentachrome staining substantiated these results. Nonetheless, the fiber network remains intact, which could be confirmed by reflection electron microscopy analysis. CONCLUSION:: In this study, we developed a procedure that grants successful decellularization of porcine aortic wall while maintaining the fibrous microstructure. We highlighted the significant effect of DNase and α-galactosidase treatment. In addition, we could show the need for a multifactorial treatment and comprehensive evaluation protocols for thick tissue decellularization.
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Aorta/citología , Aorta/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Animales , Aorta/metabolismo , Ácido Desoxicólico/farmacología , Desoxirribonucleasas/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Glicosaminoglicanos/metabolismo , Dodecil Sulfato de Sodio/farmacología , Sonicación , Porcinos , Técnicas de Cultivo de Tejidos , Andamios del Tejido , alfa-Galactosidasa/farmacologíaRESUMEN
Transcatheter aortic valve implantation (TAVI) is a fast-growing, exciting field of invasive therapy. During the last years many innovations significantly improved this technique. However, the prostheses are still associated with drawbacks. The aim of this study was to create cell-seeded biohybrid aortic valves (BAVs) as an ideal implant by combination of assets of biological and artificial materials. Furthermore, the influence of TAVI procedure on tissue-engineered BAV was investigated. BAV (n=6) were designed with decellularized homograft cusps and polyurethane walls. They were seeded with fibroblasts and endothelial cells isolated from saphenous veins. Consecutively, BAV were conditioned under low pulsatile flow (500 mL/min) for 5 days in a specialized bioreactor. After conditioning, TAVI-simulation was performed. The procedure was concluded with re-perfusion of the BAV for 2 days at an increased pulsatile flow (1100 mL/min). Functionality was assessed by video-documentation. Samples were taken after each processing step and evaluated by scanning electron microscopy (SEM), immunohistochemical staining (IHC), and Live/Dead-assays. The designed BAV were fully functioning and displayed physiologic behavior. After cell seeding, static cultivation and first conditioning, confluent cell layers were observed in SEM. Additionally, IHC indicated the presence of endothelial cells and fibroblasts. A significant construction of extracellular matrix was detected after the conditioning phase. However, a large number of lethal cells were observed after crimping by Live/Dead staining. Analysis revealed that the cells while still being present directly after crimping were removed in subsequent perfusion. Extensive regions of damaged cell-layers were detected by SEM-analysis substantiating these findings. Furthermore, increased ICAM expression was detected after re-perfusion as manifestation of inflammatory reaction. The approach to generate biohybrid valves is promising. However, damages inflicted during the crimping process seem not to be immediately detectable. Due to severe impacts on seeded cells, the strategy of living TE valves for TAVI should be reconsidered.
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Válvula Aórtica/cirugía , Bioprótesis , Prótesis Valvulares Cardíacas , Ingeniería de Tejidos/métodos , Reemplazo de la Válvula Aórtica Transcatéter , Válvula Aórtica/citología , Reactores Biológicos , Células Cultivadas , Células Endoteliales/citología , Diseño de Equipo , Fibroblastos/citología , Humanos , Poliuretanos/química , Vena Safena/citología , Andamios del Tejido/químicaRESUMEN
The aim of the study was to compare the behavior of seeded cells on synthetic and natural aortic valve scaffolds during a low-flow conditioning period. Polyurethane (group A) and aortic homograft valves (group B) were consecutively seeded with human fibroblasts (FB), and endothelial cells (EC) using a rotating seeding device. Each seeding procedure was followed by an exposure to low pulsatile flow in a dynamic bioreactor for 5 days. For further analysis, samples were taken before and after conditioning. Scanning electron microscopy showed confluent cell layers in both groups. Immunohistochemical analysis showed the presence of EC and FB before and after conditioning as well as the establishment of an extracellular matrix (ECM) during conditioning. A higher expression of ECM was observed on the scaffolds' inner surface. Real-time polymerase chain reaction showed higher inflammatory response during the conditioning of homografts. Endothelialization caused a decrease in inflammatory gene expression. The efficient colonization, the establishment of an ECM, and the comparable inflammatory cell reaction to the scaffolds in both groups proved the biocompatibility of the synthetic scaffold. The newly developed bioreactor permits conditioning and cell adaption to shear stress. Therefore, polyurethane valve scaffolds may offer a new option for aortic valve replacement.
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Válvula Aórtica/citología , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Prótesis Valvulares Cardíacas , Ingeniería de Tejidos/métodos , Reactores Biológicos , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Poliuretanos , Diseño de Prótesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Acondicionamiento Pretrasplante , Trasplante HomólogoRESUMEN
BACKGROUND: Tissue engineering represents a promising new method for treating heart valve diseases. The aim of this study was evaluate the importance of conditioning procedures of tissue engineered polyurethane heart valve prostheses by the comparison of static and dynamic cultivation methods. METHODS: Human vascular endothelial cells (ECs) and fibroblasts (FBs) were obtained from saphenous vein segments. Polyurethane scaffolds (n = 10) were primarily seeded with FBs and subsequently with ECs, followed by different cultivation methods of cell layers (A: static, B: dynamic). Group A was statically cultivated for 6 days. Group B was exposed to low flow conditions (t1=3 days at 750 ml/min, t2=2 days at 1100 ml/min) in a newly developed conditioning bioreactor. Samples were taken after static and dynamic cultivation and were analyzed by scanning electron microscopy (SEM), immunohistochemistry (IHC), and real time polymerase chain reaction (RT-PCR). RESULTS: SEM results showed a high density of adherent cells on the surface valves from both groups. However, better cell distribution and cell behavior was detected in Group B. IHC staining against CD31 and TE-7 revealed a positive reaction in both groups. Higher expression of extracellular matrix (ICAM, Collagen IV) was observed in Group B. RT- PCR demonstrated a higher expression of inflammatory Cytokines in Group B. CONCLUSION: While conventional cultivation method can be used for the development of tissue engineered heart valves. Better results can be obtained by performing a conditioning step that may improve the tolerance of cells to shear stress. The novel pulsatile bioreactor offers an adequate tool for in vitro improvement of mechanical properties of tissue engineered cardiovascular prostheses.