Ricardo Moreira

Multiple-Step Injection Molding for Fibrin-Based Tissue-Engineered Heart Valves.

Heart valves are elaborate and highly heterogeneous structures of the circulatory system. Despite the well accepted relationship between the structural and mechanical anisotropy and the optimal function of the valves, most approaches to create tissue-engineered heart valves (TEHVs) do not try to mimic this complexity and rely on one homogenous combination of cells and materials for the whole construct. The aim of this study was to establish an easy and versatile method to introduce spatial diversity into a heart valve fibrin scaffold. We developed a multiple-step injection molding process that enables the fabrication of TEHVs with heterogeneous composition (cell/scaffold material) of wall and leaflets without the need of gluing or suturing components together, with the leaflets firmly connected to the wall. The integrity of the valves and their functionality was proved by either opening/closing cycles in a bioreactor (proof of principle without cells) or with continuous stimulation over 2 weeks. We demonstrated the potential of the method by the two-step molding of the wall and the leaflets containing different cell lines. Immunohistology after stimulation confirmed tissue formation and demonstrated the localization of the different cell types. Furthermore, we showed the proof of principle fabrication of valves using different materials for wall (fibrin) and leaflets (hybrid gel of fibrin/elastin-like recombinamer) and with layered leaflets. The method is easy to implement, does not require special facilities, and can be reproduced in any tissue-engineering lab. While it has been demonstrated here with fibrin, it can easily be extended to other hydrogels.

Tissue-Engineered Fibrin-Based Heart Valve with Bio-Inspired Textile Reinforcement

The mechanical properties of tissue-engineered heart valves still need to be improved to enable their implantation in the systemic circulation. The aim of this study is to develop a tissue-engineered valve for the aortic position - the BioTexValve - by exploiting a bio-inspired composite textile scaffold to confer native-like mechanical strength and anisotropy to the leaflets. This is achieved by multifilament fibers arranged similarly to the collagen bundles in the native aortic leaflet, fixed by a thin electrospun layer directly deposited on the pattern. The textile-based leaflets are positioned into a 3D mould where the components to form a fibrin gel containing human vascular smooth muscle cells are introduced. Upon fibrin polymerization, a complete valve is obtained. After 21 d of maturation by static and dynamic stimulation in a custom-made bioreactor, the valve shows excellent functionality under aortic pressure and flow conditions, as demonstrated by hydrodynamic tests performed according to ISO standards in a mock circulation system. The leaflets possess remarkable burst strength (1086 mmHg) while remaining pliable; pronounced extracellular matrix production is revealed by immunohistochemistry and biochemical assay. This study demonstrates the potential of bio-inspired textile-reinforcement for the fabrication of functional tissue-engineered heart valves for the aortic position.

TexMi: development of tissue-engineered textile-reinforced mitral valve prosthesis.

Mitral valve regurgitation together with aortic stenosis is the most common valvular heart disease in Europe and North America. Mechanical and biological prostheses available for mitral valve replacement have significant limitations such as the need of a long-term anticoagulation therapy and failure by calcifications. Both types are unable to remodel, self-repair, and adapt to the changing hemodynamic conditions. Moreover, they are mostly designed for the aortic position and do not reproduce the native annular-ventricular continuity, resulting in suboptimal hemodynamics, limited durability, and gradually decreasing ventricular pumping efficiency. A tissue-engineered heart valve specifically designed for the mitral position has the potential to overcome the limitations of the commercially available substitutes. For this purpose, we developed the TexMi, a living textile-reinforced mitral valve, which recapitulates the key elements of the native one: annulus, asymmetric leaflets (anterior and posterior), and chordae tendineae to maintain the native annular-ventricular continuity. The tissue-engineered valve is based on a composite scaffold consisting of the fibrin gel as a cell carrier and a textile tubular structure with the twofold task of defining the gross three-dimensional (3D) geometry of the valve and conferring mechanical stability. The TexMi valves were molded with ovine umbilical vein cells and stimulated under dynamic conditions for 21 days in a custom-made bioreactor. Histological and immunohistological stainings showed remarkable tissue development with abundant aligned collagen fibers and elastin deposition. No cell-mediated tissue contraction occurred. This study presents the proof-of-principle for the realization of a tissue-engineered mitral valve with a simple and reliable injection molding process readily adaptable to the patients anatomy and pathological situation by producing a patient-specific rapid prototyped mold.

A novel small-caliber bacterial cellulose vascular prosthesis: production, characterization, and preliminary in vivo testing

Vascular grafts are used to bypass damaged or diseased blood vessels. Bacterial cellulose (BC) has been studied for use as an off-the-shelf graft. Herein, we present a novel, cost-effective, method for the production of small caliber BC grafts with minimal processing or requirements. The morphology of the graft wall produced a tensile strength above that of native vessels, performing similarly to the current commercial alternatives. As a result of the production method, the luminal surface of the graft presents similar topography to that of native vessels. We have also studied the in vivo behavior of these BC graft in order to further demonstrate their viability. In these preliminary studies, 1 month patency was achieved, with the presence of neo-vessels and endothelial cells on the luminal surface of the graft.

Effect of reinforcement volume fraction and orientation on a hybrid tissue engineered aortic heart valve with a tubular leaflet design

Transcatheter aortic valve implantation of fibrin-based tissue engineered heart valves with a tubular leaflet construct have been developed as an alternative to invasive traditional surgical heart valve implantation. In general, they are well suited for the pulmonary position, but display insufficient mechanical properties for the aortic position. To enable the application of tissue-engineered valves in the systemic circulation, the tissue is reinforced with a textile scaffold. The current study seeks to compare the effect of varying the fiber volume fraction and orientation of bidirectional textile reinforcement on the closed-valve configuration. An anisotropic large deformation material model based on structural tensors was chosen and the materials were characterized. A finite element model was constructed of the heart valve, and the pinching and suturing of the corners along with application of pressure was simulated. Virtual experiments were conducted with fiber volume fractions of 0.1, 0.01, 0.001, and 0.0001 for ±45° fiber orientations. Furthermore, volume fraction was held at 0.01 and fiber orientations of 0°, ±15°, ±30°, ±45°, ±60°, ±75° and 90° from the tube’s axial direction were simulated and compared. It was shown that increasing the fiber volume fraction decreased the maximum principle strain in the valve, but lead to less closure. Additionally, the effect of fiber orientation affected the strains differently at different locations, depending on the local deformed geometry. This indicates that a non-uniform fiber distribution using tailored fiber placement could be used to optimize reinforcement design.

Ultrasound for in vitro noninvasive, real time monitoring and evaluation of tissue-engineered heart valves.

Tissue-engineered heart valves are developed in bioreactors where biochemical and mechanical stimuli are provided for extracellular matrix formation. During this phase, the monitoring possibilities are limited by the need to maintain the sterility and integrity of the valve. Therefore, noninvasive and nondestructive techniques are required. As such, optical imaging is commonly used to verify valves functionality in vitro. It provides important information (i.e., leaflet symmetry, geometric orifice area, and closing and opening times), which is, however, usually limited to a singular view along the central axis from the outflow side. In this study, we propose ultrasound as a monitoring method that, in contrast to established optical imaging, can assess the valve from different planes, scanning the whole three-dimensional geometry. We show the potential benefits associated with the application of ultrasound to bioreactors, in advancing heart valve tissue engineering from design to fabrication and in vitro maturation. Specifically, we demonstrate that additional information, otherwise unavailable, can be gained to evaluate the valves functionality (e.g., coaptation length, and effective cusp height and shape). Furthermore, we show that Doppler techniques provide qualitative visualization and quantitative evaluation of the flow through the valve, in real time and throughout the whole in vitro fabrication phase.

Artificial Textile Reinforced Tubular Aortic Heart Valves—Multi-scale Modelling and Experimental Validation

Tissue engineered valvular implants are in development as living and remodelling prostheses to replace damaged native valves. To improve the mechanical properties of the valve, textile is used as a reinforcing scaffold. To predict the behaviour and optimize the structure of such composites, it is necessary to understand the behaviour of the underlying components. The current study seeks to test a multi-scale approach often used in the field of composites to evaluate the behaviour of knitted textile reinforced elastomeric composites. The complex textile structure is divided into simplified models at different levels/structural units. Virtual experiments are conducted at each of these levels and their responses are fit to appropriate isotropic and anisotropic hyperelastic material models. The simulation responses obtained by conducting virtual experiments on the repeating unit cell (RUC) of the composite are then compared with experimental results, resulting in good agreement. After experimental validation, the multi-scale approach is used to predict the behaviour of artificial heart valves.

Umbilical cord as human cell source for mitral valve tissue engineering - venous vs. arterial cells

Abstract Around 2% of the population in developed nations are affected by mitral valve disease and available valvular replacements are not designed for the atrioventricular position. Recently our group developed the first tissue-engineered heart valve (TEHV) specifically designed for the mitral position – the TexMi valve. The valve recapitulates the main components of the native valve, i.e. annulus, asymmetric leaflets and the crucial chordae tendineae. In the present study, we evaluated the human umbilical cord as a clinically applicable cell source for the TexMi valve. Valves produced with cells isolated from human umbilical cord veins (HUVs) and human umbilical cord arteries (HUAs) were conditioned for 21 days in custom-made bioreactors and evaluated in terms of extracellular matrix (ECM) composition and mechanical properties. In addition, static cell-laden fibrin discs were molded to investigate cell-mediated tissue contraction and differences in ECM production. HUA and HUV cells were able to deliver functional valves with a rich ECM composed mainly of collagen. Particularly noteworthy was the synthesis of elastin, which has been observed rarely in TEHV. The elastin synthesis was significantly higher in TexMi valves produced with HUV cells and therefore the HUV is considered to be the preferred cell source.