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

Abstract

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

“…In this study, we exploited the versatility of fibrin as a scaffold material and of the fibrin‐based moulding technique . The leaflets were positioned in a 3D mould and the components of the fibrin gel carefully injected in a fabrication process that resulted in complete semilunar heart valves without the need of any further procedure (e.g., suturing) which might create stiffness or calcification points .…”

“…The generally adopted approach to fabricate TEHVs is to mimic the semilunar shape of the native leaflets in the attempt to recreate the physiological haemodynamic conditions . However, due to the complex design and the insufficient mechanical properties, such valves are not capable of withstanding the harsh conditions of the systemic circulation and their application has been limited to the pulmonary position in preclinical studies …”

The future of heart valve replacement: recent developments and translational challenges for heart valve tissue engineering

“…So far, scientists have not been able to create synthetic matrices with the same unique functional characteristics and anisotropic microstructure of the native valve (e.g., flexible and nonresisted motion in systole and durable load bearing behaviour in diastole), obtaining TEHVs inadequate as aortic replacements. Recently, thanks to the introduction of the tubular leaflet approach, other groups investigated innovative fibrin‐based tissues that showed good in vitro functionality (Reimer et al , ; Weber et al , ) and, more recently, sufficient in vivo functionality up to 24 weeks in the systemic circulation of sheep with almost complete cell repopulation (Syedain et al , ).…”

“…Ideally, the advantages of minimally-invasive techniques should be combined with the innovative and promising in situ heart valve TE approach. Recent studies investigated the possibility of merging the transcatheter techniques with living and off-the-shelf decellularized TEHVs, showing the feasibility of this approach in vitro and in vivo, in sheep (Dijkman et al, 2012b;Driessen-Mol et al, 2014) and baboon (Weber et al, 2011(Weber et al, , 2015 models. These preliminary results suggest the potential to significantly improve current treatment options for patients suffering from VHD, in particular when the most advanced implantation techniques and devices are used.…”

“…Recently, thanks to the introduction of the tubular leaflet approach, other groups investigated innovative fibrinbased tissues that showed good in vitro functionality Weber et al, 2015) and, more recently, sufficient in vivo functionality up to 24 weeks in the systemic circulation of sheep with almost complete cell repopulation .…”