Transcatheter heart valve substitutes are rapidly evolving as alternative treatment to surgical valve replacement.5,40 Nevertheless, current clinically available bioprostheses for TPVR suffer from major drawbacks such as continuous degeneration and structural deterioration, making them inadequate, especially for young adults and child patients. To address this increasing clinical need, TEHVs have been proposed as a potential solution, being characterized by properties such as long-term durability and regeneration capacities.5,40

Decellularized in vitro grown TEM-based valves have been successfully applied as TPVR in numerous preclinical studies in non-human primate and ovine models.16,17,18,19,20,21,22 Recently, their long-term durability, remodeling potential, and clinical relevance have been highlighted in a successful 1-year follow-up study.18 Nevertheless, xenogeneic cell sources have been often employed to produce the TEM, hampering the clinical translation of such prostheses. Recent investigations evaluated the functionality and remodeling capacities of non-fixed decellularized xenogeneic heart valves. After promising preclinical results,41,42 such prostheses have advanced into a clinical setting and were implanted in pediatric patients, resulting in severe immune reactions and fatal failure of the prostheses,32 thus raising many concerns on their safety.33 Therefore, the transition of culture protocols from animal-derived to human-derived cell sources is of pivotal importance for the safe translation of TEHVs into clinical practice.

In this context, the production of a robust human cell-derived TEM is fundamental to ensure immediate functionality of the TEHVs exposed to the hemodynamic loading. Here, we developed new culture protocols using hDF, to produce a compact and organized hTEM. Other groups previously investigated the potential of hDF for cardiovascular TE applications.24,37,43 These studies showed promising results in terms of functionality and host response, including recellularization and absence of calcifications.

Based on this promising data, we here further developed in vitro culture protocols for TEHVs with increased translational potential.

With the addition of TGF-β1, we stimulated collagen production and increased tissue organization in vitro.44,45 TGF-β1 is a well-known cytokine involved in several physiological processes like valvulogenesis,46 valve repair,47 cell proliferation, and ECM production.44,45 Our results suggested that the supplementation of 5 ng/ml of TGF-β1 led to increased GAGs production, comparable HYP content, and a qualitatively denser and more reproducible collagenous matrix in comparison with the other tested groups. Importantly, by extending the in vitro culture time, it will be possible to obtain a more robust matrix capable to sustain the aortic pressure conditions.19

Clinical translation of tissue-engineered products is challenged by many factors. To date, the large heterogeneity of culture methods reported in literature, the risks of infection during culture, the non-automated and non-scalable procedures, and the disregard of xeno-free protocols still limit the advancement of TEHVs to a clinical setting.48,49 However, although they are not yet clinically approved in the setting of heart valves, small caliber conduits produced with this approach, have already been advanced into clinical application providing safe and functional results.40,50,51 To hasten bench-to-bedside translation, it is necessary to establish Good Manufacturing and Good Laboratory Practices (GMP, GLP) procedures. The development of standardized manufacturing processes and quality control systems, both compliant with the International Organization for Standardization (ISO), have to be considered in order to satisfy the technical and infrastructural requirements for the commercialization of TEHVs.48,52

In addition, valve design has been recognized to be a key factor in order to achieve TEHVs long-term functionality and positive remodeling.18,28,29,30 In this regard, new computational modeling studies have recently underlined that physiological-like pre-shaping of TEHVs during in vitro culture may be a key player for the long-term durability and functionality of such substitutes.18,28,29 Here, we further enhanced this concept by introducing another fundamental anatomical feature of the native valves: the Valsalva sinuses.

The heart valve sinuses have the physiological role of actively promote and influence the opening and closing behavior of the leaflets, generate blood vortices, and consequently blood wash-out during diastole.35,36 Through this mechanism, the Valsalva sinuses are responsible for the decreased mechanical stress and strain sensed by the leaflets, thereby distributing the hemodynamic loading and preventing radial cusps contraction.28,36,53,54,55,56

From a clinical point of view, the importance of the Valsalva sinuses has been described in several surgical preservation and reconstruction procedures showing the benefits that such anatomical features bring to the patients.57,58,59 Despite this knowledge, clinically available transcatheter bioprostheses do not integrate Valsalva sinuses in their design.

Hence, based on a previously reported study from our group18 as well as others,36,55,60,61 we hypothesized that by implementing the Valsalva sinuses in our initial valve geometry, we could favor valve functionality and its future remodeling.

So far, only two studies have reported the implementation of Valsalva sinuses into TEHVs.20,62 Dodge–Khatami and colleagues62 used decellularized equine valved jugular veins as TPVR in sheep, showing promising functionality and endothelialization potential. However, the use of non-fixed xenogeneic material may represent a significant limitation for their future clinical translation.

In a recent proof-of-concept study, we demonstrated the technical feasibility of using sinus-stented-TEHVs as TPVR and showed their functionality in a short-term follow-up period (up to 16 weeks).20 However, owing to a suboptimal initial valve design, leaflet retraction and valve insufficiency occurred over time.

To overcome this limitation, in the present study, we have modified the valve design by (i) implementing physiological belly-shape in the leaflets, (ii) increasing the coaptation area, and (iii) using a different suturing pattern to integrate the scaffold into the stent prior to in vitro culture. In addition, the hTESVs described here, were manufactured with a human cell source, thereby enhancing their clinical relevance. This resulted in successful in vitro valve testing and in vivo functionality, with optimal valve performance up to 4 hours follow-up in a translational FDA-recommended ovine model without evidence of regurgitation or stenosis. Chronic studies are however needed to confirm the remodeling potential of the newly developed hTEM as well as the long-term functionality of the prosthesis.

Our study has several limitations. First, the number of animals used in this study was limited, but sufficient to prove the feasibility of our approach. Second, short- (e.g., 1–3 months) and long-term (≥6 months) follow-up are fundamental in order to assess valve functionality and remodeling over time, even if the remodeling potential of a similar matrix was previously confirmed by others.23,24 Compared with our previous work,20 major changes (i.e., different cell source, different valve and leaflet geometry) were applied to obtain the hTESV and, therefore, the novel valve design needed to be tested in an acute feasibility study. Although chronic experiments were beyond the scope of this study, they should be addressed in the future. Third, the stress distribution on the leaflets should be validated with computational modeling tools, whereas the flow patterns determined by the implemented Valsalva sinuses should be confirmed using flow magnetic resonance imaging technologies.

In conclusion, in this study we demonstrated the possibility to develop a human cell-derived TEM for heart valve tissue engineering applications. In addition, we proposed a novel valve design, which includes the anatomical feature of the Valsalva sinuses, and we showed the feasibility and safety of TPVR using an off-the-shelf available human cell-derived TESVs in an ovine model.