Pulmonary valve tissue engineering strategies in large animal models

In the last 25 years, numerous tissue engineered heart valve (TEHV) strategies have been studied in large animal models. To evaluate, qualify and summarize all available publications, we conducted a systematic review and meta-analysis. We identified 80 reports that studied TEHVs of synthetic or natural scaffolds in pulmonary position (n = 693 animals). We identified substantial heterogeneity in study designs, methods and outcomes. Most importantly, the quality assessment showed poor reporting in randomization and blinding strategies. Meta-analysis showed no differences in mortality and rate of valve regurgitation between different scaffolds or strategies. However, it revealed a higher transvalvular pressure gradient in synthetic scaffolds (11.6 mmHg; 95% CI, [7.31–15.89]) compared to natural scaffolds (4,67 mmHg; 95% CI, [3,94–5.39]; p = 0.003). These results should be interpreted with caution due to lack of a standardized control group, substantial study heterogeneity, and relatively low number of comparable studies in subgroup analyses. Based on this review, the most adequate scaffold model is still undefined. This review endorses that, to move the TEHV field forward and enable reliable comparisons, it is essential to define standardized methods and ways of reporting. This would greatly enhance the value of individual large animal studies.

Developing the Tissue Engineered Heart Valve – a Descriptive Hemodynamic and Ultrasound in Vitro Characterization Study of Heart Valves in a Bioreactor

The inherent limitations of current heart valve substitutes create the premise for the Tissue Engineered Heart Valve (TEHV), considered the perfect substitute. We aimed to compare in vitro hemodynamic performances of our TEHV, the conventional prosthetic valve and similar porcine valves, by ultrasonography and geometry resulting in six valve models analysis. In a bioreactor, pulmonary and aortic physiology were replicated thus hemodynamic characteristics were tested. Using ultrasound, transvalvular pressure gradients and flow were measured and used to calculate their valvular functional area (VFA) and using a high-speed camera, the geometric peak opening area (GOA) was assessed. The obtained results were normalized to the diameter of the biological prosthesis in order to increase the measurement’s accuracy. The ultrasound revealed normal function of all valves and physiologic transvalvular pressure gradients. The TEHV scaffold revealed absence of laceration or dehiscence, and performances in accordance with the control prostheses. The GOA was facile to obtain and the normalized values proved to be greater than the calculated functional area in all analyzed cases and the peak opening areas resulted lesser for the aortic conditions for all six used valves prototypes. To our knowledge, this is the first study to use bioreactors, for in vitro evaluation of heart valves.

Mechanical and Degradation Properties of Hybrid Scaffolds for Tissue Engineered Heart Valve (TEHV)

In addition to biocompatibility, an ideal scaffold for the regeneration of valvular tissue should also replicate the natural heart valve extracellular matrix (ECM) in terms of biomechanical properties and structural stability. In our previous paper, we demonstrated the development of collagen type I and hyaluronic acid (HA)-based scaffolds with interlaced microstructure. Such hybrid scaffolds were found to be compatible with cardiosphere-derived cells (CDCs) to potentially regenerate the diseased aortic heart valve. This paper focused on the quantification of the effect of crosslinking density on the mechanical properties under dry and wet conditions as well as degradation resistance. Elastic moduli increased with increasing crosslinking densities, in the dry and wet state, for parent networks, whereas those of interlaced scaffolds were higher than either network alone. Compressive and storage moduli ranged from 35 ± 5 to 95 ± 5 kPa and 16 ± 2 kPa to 113 ± 6 kPa, respectively, in the dry state. Storage moduli, in the dry state, matched and exceeded those of human aortic valve leaflets (HAVL). Similarly, degradation resistance increased with increasing the crosslinking densities for collagen-only and HA-only scaffolds. Interlaced scaffolds showed partial degradation in the presence of either collagenase or hyaluronidase as compared to when exposed to both enzymes together. These results agree with our previous findings that interlaced scaffolds were composed of independent collagen and HA networks without crosslinking between them. Thus, collagen/HA interlaced scaffolds have the potential to fill in the niche for designing an ideal tissue engineered heart valve (TEHV).

Strategies to Improve Survival from Surgery for Heart Valve Implantation in Sheep

Sheep are a commonly used and validated model for cardiovascular research and, more specifically, for heart valve research.Implanting a heart valve on the arrested heart in sheep is complex and is often complicated by difficulties in restarting the heart, causing significant on-table mortality. Therefore, optimal cardioprotective management during heart valve implantation in sheep is essential. However, little is known about successful cardioprotective management techniques in sheep. This article reports our experience in the cardioprotective management of 20 female sheep that underwent surgical aortic valve replacement with a stented tissue-engineered heart valve prosthesis. During this series of experiments, we modified our cardioprotection protocol to improve survival. We emphasize the importance of total body hypothermia and external cooling of the heart. Furthermore, we recommend repeated cardioplegia administration at 20 min intervals during surgery, with the final dosage of cardioplegia given immediately before the de-clamping of the aorta. To reduce the number of defibrillator shocks during a state of ventricular fibrillation (VF), we have learned to restart the heart by reclamping the aorta, administeringcardioplegia until cardiac arrest, and de-clamping the aorta thereafter. Despite these encouraging results, more researchis needed to finalize a protocol for this procedure.

The VALVE REGEN project. Developing an innovative tissue engineered heart valve, in vitro and in vivo testing – preliminary results

Background Regenerative Medicine and Tissue Engineering are the grounds on which multidisciplinary teams aspire to obtain the perfect valvular substitute, which overcomes shortcomings of the present prostheses. Purpose Was to obtain a tissue engineered heart valve (TEHV) by repopulating with valvular resident cells – endothelial (EC) and fibroblasts (FB) a decellularized heart valve scaffold. Then their functionality and behavior was assessed in vitro and in vivo. Methods This study is part of a research grant approved by the Ethics Committee of the University. Six ovine pulmonary valves underwent a perfusion based decellularization protocol. Using a sequence of chemical and enzymatic treatment under a pressure gradient, cell removal was achieved and attested by histological investigations (DAPI nuclear staining –4',6-diamidino-2-phenylindol and haematoxylin-eosin) and DNA extraction. Ovine sub-dermal adipose tissue was harvested followed by stem cells isolation and culture. Using Endothelial Cell Growth Supplement and mechanical stimuli EC were differentiated and with Transforming Growth Factor-B1, FB were obtained. FB were internally seeded into cuspis bases using a 22 gauge needle and externally on the adventitia by using a rotator allowing a uniform distribution and seeding of cells. EC were seeded into leaflets pockets and intra-luminal also using the rotator. The repopulated valves were preconditionated in a bioreactor by gradually exposing them to the pulmonary hemodynamic regimen. By using a high speed camera, their behavior was examined when exposed to in vivo conditions. The in vivo testing was performed by surgical implantation in the gold model considered animal – the sheep. By transesophageal ultrasound (TEE US) and epicardic US, their intra-operatory function was evaluated. Post-procedure, evaluation was performed by periodic trans-thoracic (TTE US). Results Six TEHV were obtained. The decellularization histology assessment revealed acellular scaffolds and non-detectable nucleic material at the DNA extraction. Six adipose derived stem cells cultures were obtained and subsequently specialized towards EC and FB lines. The repopulation procedures underwent without incidents. During the bioreactor preconditioning, the TEHV showed complete opening and competent central coaptation. Leaflets presented physiological movement and absence of damage of valvular apparatus. The TEE US evaluation in vivo revealed normal valvular function without signs of stenosis or regurgitation. The periodic TTE US showed preserved valvular function. Conclusions Our preliminary results point out a manufactured TEHV with physiological behavior when tested in vitro and in vivo. Their interaction with a living body will be pointed out only in the explant phase, after histology analysis. The present results appear optimistic but only extended studies and follow-ups will certify their superiority in terms of performances and behavior. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This paper was financed by a grant from the Competitiveness Operational Programme 2014-2020, Tissue engineering technologies for cardiac valve regeneration, valve-regen, id P_37_673, Mysmis code: 103431, contract 50/05.09.2016.