Tissue Engineered Heart Valve Constructs Research Articles

The Jagged-1/Notch1 Signaling Pathway Promotes the Construction of Tissue-Engineered Heart Valves.

Background: Tissue-engineered heart valves (TEHVs) are promising new heart valve substitutes for valvular heart disease. The Notch signaling pathway plays a critical role in the development of congenital heart valves. Objective: To investigate the role of the Notch signaling pathway in the construction of TEHVs. Methods: The induced endothelial cells, which act as seed cells, were differentiated from adipose-derived stem cells and were treated with Jagged-1 (JAG-1) protein and γ-secretase inhibitor (DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester), respectively. Cell phenotypic changes, the expression of proteins relating to the epithelial-mesenchymal transition (EMT), and changes in paxillin expression were detected. Decellularized valve scaffolds were produced from decellularized porcine aortic valves. The seed cells were them inoculated into Matrigel-coated flap scaffolds for complex culture and characterization. Results: JAG-1 significantly reduced apoptosis and promoted the seeded cells' proliferation and migration ability, in contrast to the treatment of DAPT. In addition, the expression of EMT-related proteins, E-cadherin and N-cadherin, was significantly increased after treatment with JAG-1 and was reduced after the application of DAPT. Meanwhile, the adhesive-related expression of paxillin and fibronectin proteins was increased after the activation of Notch1 signaling and vice versa. Of interest, activation of the Notch1 signaling pathway resulted in more closely arranged cells on the valve surface after recellularization. Conclusion: Activation of the JAG-1/Notch1 signaling pathway increased seeded cells' proliferation and migratory ability and promoted the EMT and adhesion of seed cells, which was conducive to binding to the matrix, facilitating accelerated endothelialization of TEHVs.

Abstract 339: Hematopoietic-derived Progenitor Cells Identified in Mouse Heart Valves are also Present in Human Mitral Valves

Tissue engineered heart valve constructs (TEHVCs) are thought to be the solution for the increasing needs of heart valve replacements. Using decellularized xenograft valve matrices reseeded with autologous cells seems to fulfill the demands for durability, lack of coagulation and immune reactions, in the same time allowing growth and adaptability. The presently used cell sources are not viable as the TEHVCs frequently exhibit durability issues due to inefficient seeding and subsequent calcification. The natural valve interstitial cells are very heterogeneous, their role is to maintain the extracellular matrix which is responsible for bearing the mechanical and hemodynamic load. We have previously identified, in murine heart valves, a hematopoietic bone marrow-derived progenitor cell population. These progenitor cells are continuously migrating into the heart valves and appear to participate in the normal homeostasis. The heart valve progenitor cells identified in mice seems a good candidate for TEHVCs, but first, their existence must be validated in human heart valves. We used commercially available, paraffin-embedded, human mitral valve sections from 4 different individuals to immunohistochemically identify the hematopoietic-derived progenitor cells. Combination of anti-CD45 (pan hematopoietic marker) and anti-CD133 (progenitor marker) antibodies revealed a double-positive cell population in the proximal region (toward the annulus), atrial side of the mitral leaflets - exactly where we have found the similar cells in the murine system. By further phenotypic experiments, we have excluded the presence of B- and T-lymphocytes, granulocytes and macrophages. In contrast to the mouse findings, the human CD45 + /CD133 + cells are also expressing CD34 which further emphasizes their hematopoietic origin (in mouse the CD34 is not a hematopoietic marker). By co-labeling with CD31 (Pecam-1) markers, we found that the CD45 + /CD133 + cells are never part of the endocardium, although they can be observed closely underneath. In conclusion, we have identified the CD45 + /CD133 + cells, previously identified in mice as progenitor cells, in human mitral valves. Functional characterization of these cells and their use in TEHVCs is the focus of our future work.

Biomechanical properties of native and tissue engineered heart valve constructs

Due to the increasing number of heart valve diseases, there is an urgent clinical need for off-the-shelf tissue engineered heart valves. While significant progress has been made toward improving the design and performance of both mechanical and tissue engineered heart valves (TEHVs), a human implantable, functional, and viable TEHV has remained elusive. In animal studies so far, the implanted TEHVs have failed to survive more than a few months after transplantation due to insufficient mechanical properties. Therefore, the success of future heart valve tissue engineering approaches depends on the ability of the TEHV to mimic and maintain the functional and mechanical properties of the native heart valves. However, aside from some tensile quasistatic data and flexural or bending properties, detailed mechanical properties such as dynamic fatigue, creep behavior, and viscoelastic properties of heart valves are still poorly understood. The need for better understanding and more detailed characterization of mechanical properties of tissue engineered, as well as native heart valve constructs is thus evident. In the current review we aim to present an overview of the current understanding of the mechanical properties of human and common animal model heart valves. The relevant data on both native and tissue engineered heart valve constructs have been compiled and analyzed to help in defining the target ranges for mechanical properties of TEHV constructs, particularly for the aortic and the pulmonary valves. We conclude with a summary of perspectives on the future work on better understanding of the mechanical properties of TEHV constructs.

Reendothelialization of Human Heart Valve Neoscaffolds Using Umbilical Cord-Derived Endothelial Cells

Heart valve tissue engineering represents a concept for improving the current methods of valvular heart disease therapy. The aim of this study was to develop tissue engineered heart valves combining human umbilical vein endothelial cells (HUVECs) and decellularized human heart valve matrices. Pulmonary (n=9) and aortic (n=6) human allografts were harvested from explanted hearts from heart transplant recipients and were decellularized using a detergent-based cell extraction method. Analysis of decellularization success was performed with light microscopy, transmission electron microscopy and quantitative analysis of collagen and elastin content. The decellularization method resulted in full removal of native cells while the mechanical stability and the quantitative composition of the neoscaffolds was maintained. The luminal surface of the human matrix could be successfully recellularized with in vitro expanded HUVECs under dynamic flow conditions. The surface appeared as a confluent cell monolayer of positively labeled cells for von Willebrand factor and CD 31, indicating their endothelial nature. Human heart valves can be decellularized by the described method. Recellularization of the human matrix resulted in the formation of a confluent HUVEC monolayer. The in vitro construction of tissue-engineered heart valves based on decellularized human matrices followed by endothelialization using HUVECs is a feasible and safe method, leading to the development of future clinical strategies in the treatment of heart valve disease.

Variation in Tissue Outcome of Ovine and Human Engineered Heart Valve Constructs: Relevance for Tissue Engineering

Clinical application of tissue engineered heart valves requires precise control of the tissue culture process to predict tissue composition and mechanical properties prior to implantation, and to understand the variation in tissue outcome. To this end we investigated cellular phenotype and tissue properties of ovine (n = 8) and human (n = 7) tissue engineered heart valve constructs to quantify variations in tissue outcome within species, study the differences between species and determine possible indicators of tissue outcome. Tissue constructs consisted of polyglycolic acid/poly-4-hydroxybutyrate scaffolds, seeded with myofibroblasts obtained from the jugular vein (sheep) or the saphenous vein (from humans undergoing cardiac surgery) and cultured under static conditions. Prior to seeding, protein expression of α-smooth muscle actin, vimentin, nonmuscle myosin heavy chain and heat shock protein 47 were determined to identify differences at an early stage of the tissue engineering process. After 4 weeks of culture, tissue composition and mechanical properties were quantified as indicators of tissue outcome. After 4 weeks of tissue culture, tissue properties of all ovine constructs were comparable, while there was a larger variation in the properties of the human constructs, especially the elastic modulus and collagen content. In addition, ovine constructs differed in composition from the human constructs. An increased number of α-smooth muscle actin-positive cells before seeding was correlated with the collagen content in the engineered heart valve constructs. Moreover, tissue stiffness increased with increasing collagen content. The results suggest that the culture process of ovine tissues can be controlled, whereas the mechanical properties, and hence functionality, of tissues originating from human material are more difficult to control. On-line evaluation of tissue properties during culture or more early cellular markers to predict the properties of autologous tissues cultured for individual patients are, therefore, of utmost importance for future clinical application of autologous heart valve tissue engineering. As an example, this study shows that α-smooth muscle actin might be an indicator of tissue mechanical properties.

Aging Adversely Impacts Biological Properties of Human Bone Marrow‐derived Mesenchymal Stem Cells: Implications for Tissue Engineering Heart Valve Construction

Our aim was to study the aging effects on the in vitro biological properties of bone marrow-derived mesenchymal stem cells (BMSCs) for construction of tissue-engineered heart valves. BMSCs were taken from teenagers with congenital heart diseases, and middle-aged and elderly patients with valvular diseases. Proliferative abilities were compared among the three groups by using colony-forming unit counting and growth curves (5-bromo-2'-deoxyuridine assay). Cell differentiation, vascular endothelial growth factor (VEGF) release under hypoxic condition, and migratory abilities were compared as well. Colony-forming units in the teenage group were significantly greater than those in the other two groups (P < 0.05), and significantly higher counts were observed in the middle age group than in the aged group (P < 0.05). Growth curves presented similar trends in which cells' proliferative abilities in the aged group decreased significantly (P < 0.05), while no differences were noted between the two nonaged groups. The differentiation potential to endothelial cells, osteoblasts and adipocytes, VEGF releases, and migratory abilities differed significantly between the aged group and nonaged groups (P < 0.05). However, no differences were noted between the two nonaged groups. BMSCs from older patients with heart valve diseases could be harvested and expanded successfully, and the phenotype and morphology were uniform as nonaged groups. However, the proliferative and differentiation properties of aged cells, as well as cytokine release and migratory abilities, are significantly impaired.

Endothelial Progenitor Cells as a Sole Source for Ex Vivo Seeding of Tissue-Engineered Heart Valves

We investigated whether circulating endothelial progenitor cells (EPCs) can be used as a cell source for the creation of a tissue-engineered heart valve (TEHV). Trileaflet valved conduits were fabricated using nonwoven polyglycolic acid/poly-4-hydroxybutyrate polymer. Ovine peripheral blood EPCs were dynamically seeded onto a valved conduit and incubated for 7, 14, and 21 days. Before seeding, EPCs were shown to express CD31(+), eNOS(+), and VE-Cadherin(+) but not alpha-smooth muscle actin. Histological analysis demonstrated relatively homogenous cellular ingrowth throughout the valved conduit. TEHV constructs revealed the presence of endothelial cell (EC) markers and alpha-smooth muscle actin(+) cells comparable with native valves. Protein levels were comparable with native valves and exceeded those in unseeded controls. EPC-TEHV demonstrated a temporal pattern of matrix metalloproteinases-2/9 expression and tissue inhibitors of metalloproteinase activities comparable to that of native valves. Mechanical properties of EPC-TEHV demonstrated significantly greater stiffness than that of the unseeded scaffolds and native valves. Circulating EPC appears to have the potential to provide both interstitial and endothelial functions and could potentially serve as a single-cell source for construction of autologous heart valves.

Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells

Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA). EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells. EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

Construction of Tissue-Engineered Homograft Bioprosthetic Heart Valves In Vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.

Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch

The synthesis of appropriate extracellular matrix by cells in tissue engineered heart valve constructs will be important for the maintenance of valve cusp integrity and function. We have examined and compared the capacity of mesenchymal stem cells to synthesise collagen in response to stretch in comparison with native aortic valve interstitial cells. Cells were stretched on a Flexercell FX4000 apparatus and total collagen synthesis was measured by the incorporation of [3H]-proline. The effect of stretch on gene expression of different collagen types was assessed by RT-PCR. There was a significant (p<0.01) increase in [3H]-proline incorporation into stretched valve cells at 10%, 14% and 20% stretch. The response of mesenchymal stem cells at 14% stretch was similar to that seen in the valve cells. Incorporation of [3H]-proline into soluble proteins in the cell media was significantly higher (p<0.01) only at 14% and 20% stretch in valve interstitial cells. These effects were shared with mesenchymal stem cells at 14% stretch. RT-PCR experiments demonstrated that 14% stretch up-regulated levels of mRNA for COL3A1 gene (type III collagen) but did not increase the expression of COL1A1 gene (type I collagen) in valve interstitial cells. However, both collagen genes could be detected in non-stretched and stretched mesenchymal stem cells. There was no evidence that the mesenchymal stem cells had started to adopt an osteoblastic cell phenotype in response to stretch. Collagen synthesis by valve interstitial cells is dependent upon the degree and duration of stretch. This response can be mimicked closely by exposure of mesenchymal stem cells to the same stretching profile. These properties could have important implications for the choice of cells and programme of conditioning with which to tissue engineer heart valves.