The contribution of matrix and cells to leaflet retraction in heart valve tissue engineering

Heart valve tissue engineering is a promising technique to overcome the drawbacks of currently used mechanical and prosthetic heart valve replacements. Tissue engineered (TE) heart valves are viable and autologous implants that have the capacity to grow, remodel and repair throughout a patient’s life, without the need of anticoagulation therapy. The valves are made by seeding extracellular matrix (ECM) producing cells, such as vascular-derived cells, onto a rapidly degrading scaffold material manufactured into the shape of a heart valve. TE valves are cultured constraint whereby the leaflets fuse together. During four weeks of culture, the scaffold is replaced by newly formed tissue, while stress is generated within the tissue by traction forces exerted by the cells. This stress is beneficial for tissue formation and architecture. However, during culture it causes tissue compaction, resulting in leaflet flattening, and at time of implantation, the leaflet constraints are released and the generated stress causes retraction of the leaflets. Due to this retraction, the leaflets are not able to fully close during diastole and valvular regurgitation occurs. In this thesis, this phenomenon of tissue retraction was examined and strategies to decrease retraction were investigated. The first aim was to unravel and quantify stress, compaction and retraction in developing engineered heart valve tissues. Therefore, a representative in vitro model system of rectangular TE strips (TE constructs) was developed in which the evolution of stress and compaction during culture, and the resulting retraction after release of constraints, can be quantified from a single TE construct. An important finding was that during the first 2 weeks of tissue culture, the scaffold was able to counterbalance the traction forces of the cells, which reveals a key role of the stiffness of the cellular surroundings in compaction. When scaffold degradation started after 2 weeks, stress generation and compaction became evident and gradually continued up to week 4. In average, the TE constructs compacted 50-65% in width and reached force levels of 15-55 mN and stress levels of 5-30 kPa. The resulting retraction 24 hours after release of constraints was 35-50% in length. These degrees of compaction and retraction thus seriously affect leaflet geometry and need substantial reduction to prevent regurgitation. Subsequently, the relative contributions of passive and active retraction were examined. Passive retraction occurs through passive stress release in the cells and ECM at release of constraints, while active retraction is caused by the traction forces of the cells. To quantify the active and passive contributions, the active traction forces of the cells were eliminated by Cytochalasin D or an inhibitor of the Rho-associated kinase pathway, while both passive and active contributions of the cells were eliminated by lysis and/or removal of the cells. A major finding in this study was that the passive contribution of cells to retraction is substantial. It was found that passive cell retraction accounts for 45% of total retraction, while active cell retraction accounts for 40% of the total retraction. The remaining 15% is attributed to passive retraction of the ECM. These findings illustrate the importance of the cells in the process of tissue retraction, not only actively retracting the tissue, but also in a passive manner. Finally, we aimed to decrease tissue retraction in order to obtain functional, non-retracting leaflets. As it was hypothesized that a strong and well-developed ECM would provide more resistance to the cell traction forces, two strategies to improve the mechanical integrity of the ECM were investigated. First, the effect of the environmental factor oxygen concentration on the ECM formation was investigated at both cell (2D) and tissue (3D) level. At cellular level, culturing at oxygen concentrations of 4% and below (hypoxia) enhanced the production of collagen cross-link enzymes and to a lesser extent collagen type I and III. Unfortunately, these results did not translate into enhanced collagen deposition and maturation in TE constructs. Tissue properties remained similar at 7% and 4% O2 as compared to 21% O2, while culturing below 4% O2 reduced ECM production and the mechanical integrity of the tissue. From this latter study, it was concluded that hypoxia is not very likely to create a more robust ECM. In the second strategy, the ECM integrity was hypothesized to increase by prolonged tissue culture. Collagen content and cross-linking remained constant when increasing culture time from 4 weeks to 6 and 8 weeks, but GAG content increased, which resulted in thicker tissues. Although the generated force remained constant from week 4 on, the increased thickness contributed to a decrease in generated stress. The most important finding in this study was that retraction decreased by ~50% at week 6 and 8 compared to week 4, likely due to the increased GAG content. These findings emphasize the role of the ECM in tissue retraction and that changing its composition might represent an important strategy to reduce tissue retraction and, thereby, valvular regurgitation. To summarize, solving the problem of leaflet retraction in heart valve tissue engineering remains a challenge due to the substantial contribution of passive retraction. The cellular surroundings have shown to affect the resulting compaction and retraction. However, improving resistance against cell traction forces by enhancing the compressive stiffness of the ECM has proven to be difficult. Therefore, other promising approaches like a slower degrading scaffold, adjustment of the valve geometry or decellularization of the tissue are discussed in the present thesis and will be focus of future research. Although these strategies still require extensive research, the new insights into the mechanisms of leaflet retraction obtained within this thesis provide useful knowledge needed to deal with the problem in order to develop functional tissue engineered heart valves.