An in vitro model system to quantify stress generation, compaction, and retraction in engineered heart valve tissue

\u3cp\u3eAutologous heart valve tissue engineering relies on extracellular matrix production by cells seeded into a degrading scaffold material. The cells naturally exert traction forces to their surroundings, and due to an imbalance between scaffold, tissue, and these traction forces, stress is generated within the tissue. This stress results in compaction during culture and retraction of the leaflets at release of constraints, causing shape loss of the heart valve leaflets. In the present study, an in vitro model system has been developed to quantify stress generation, compaction, and retraction during culture and after release of constraints. Tissue-engineered (TE) constructs based on polyglycolic acid/poly-4-hydroxybutyrate scaffolds seeded with human vascular-derived cells were cultured for 4 weeks. Compaction in width was measured during culture, stress generation was measured during culture and after release of constraints at week 4, and contraction was measured after release of constraints at week 4. Both compaction and stress generation started after 2 weeks of culture and continued up to week 4. TE constructs compacted up to half of their original width and reached an internal stress of 6-8kPa at week 4, which resulted in a retraction of 36%. The model system has provided a useful tool to unravel and optimize the balance between the different aspects of TE constructs to develop functional TE leaflets.\u3c/p\u3

Controlling matrix formation and cross-linking by hypoxia in cardiovascualr tissue engineering

In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O2 (n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen 1(I) and 1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O2 (n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O2. Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen 1(I) and 1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O2, LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O2). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development

Low oxygen concentrations impair tissue development in tissue-engineered cardiovascular constructs

Cardiovascular tissue engineering has shown considerable progress, but in vitro tissue conditioning to stimulate the development of a functional extracellular matrix still needs improvement. We investigated the environmental factor oxygen concentration for its potential to increase the amount of collagen and collagen cross-links, and therefore improve tissue quality. Cardiovascular tissue engineered (TE) constructs, made of rapidly degrading PGA/P4HB scaffold seeded with human vascular-derived cells, were cultured at 7%, 4%, 2%, 0.5% O2 for 4 weeks and compared to control cultures at 21% O2. Tissue properties were evaluated by measuring the extracellular matrix production and mechanical behavior. The culture environment was monitored closely and the oxygen gradient throughout the constructs was simulated with a theoretical model. TE constructs cultured at 21%, 7% and 4% O2 showed dense and homogeneous tissue formation with comparable strength, stiffness, collagen and collagen cross-link content. At 2% O2, collagen content and stiffness decreased, whereas at 0.5% O2, hardly any tissue was formed. Overall, tissue properties deteriorated at the lowest oxygen concentrations, opposing our hypothesis that was based on previous culture at low oxygen concentrations. Further research will focus on establishing the balance between applied oxygen conditions (concentration and exposure time) and optimal tissue outcome

Improved geometry of decellularized tissue engineered heart valves to prevent leaflet retraction

Recent studies on decellularized tissue engineered heart valves (DTEHVs) showed rapid host cell repopulation and increased valvular insufficiency developing over time, associated with leaflet shortening. A possible explanation for this result was found using computational simulations, which revealed radial leaflet compression in the original valvular geometry when subjected to physiological pressure conditions. Therefore, an improved geometry was suggested to enable radial leaflet extension to counteract for host cell mediated retraction. In this study, we propose a solution to impose this new geometry by using a constraining bioreactor insert during culture. Human cell based DTEHVs (n = 5) were produced as such, resulting in an enlarged coaptation area and profound belly curvature. Extracellular matrix was homogeneously distributed, with circumferential collagen alignment in the coaptation region and global tissue anisotropy. Based on in vitro functionality experiments, these DTEHVs showed competent hydrodynamic functionality under physiological pulmonary conditions and were fatigue resistant, with stable functionality up to 16 weeks in vivo simulation. Based on implemented mechanical data, our computational models revealed a considerable decrease in radial tissue compression with the obtained geometrical adjustments. Therefore, these improved DTEHV are expected to be less prone to host cell mediated leaflet retraction and will remain competent after implantation

Biomechanics and modeling of tissue-engineered heart valves

Heart valve tissue engineering (HVTE) is a promising technique to overcome the limitations of currently available heart valve prostheses. However, before clinical use, still several challenges need to be overcome. The functionality of the developed replacements is determined by their biomechanical properties and, ultimately, by their collagen architecture. Unfortunately, current techniques are often not able to induce a physiological tissue remodeling, which compromises the long-term functionality. Therefore, a deeper understanding of the process of tissue remodeling is required to optimize the phenomena involved via improving the current HVTE approaches. Computational simulations can help in this process, being a valuable and versatile tool to predict and understand experimental results. This chapter first describes the similarities and differences in functionality and biomechanical properties between native and tissue-engineered heart valves. Secondly, the current status of computational models for collagen remodeling is addressed and, finally, future directions and implications for HVTE are suggested