Since 2000, efforts have focused on developing living heart valves through tissue engineering.

Decellularized scaffolds have shown reduced immunogenicity and favorable outcomes in animal models, but the benefits of recellularization remain debated. Limited comparative studies report inconsistent results. Tudorache et al. demonstrated that seeding decellularized aortic valves with mature ECs isolated from jugular veins prevented calcification, inflammation, and degeneration, yielding excellent hemodynamic outcomes in young sheep [29]. Conversely, Theodoridis et al. reported no functional or structural improvements using ECs-like cells differentiated from mononuclear peripheral blood cells or CCN1(recombinant human pro-angiogenic factor) coating in elderly sheep, emphasizing the importance of cell source and age [30]. Seeking alternatives to conventional prostheses, Driessen-Mol et al. assessed "off-the-shelf" TEHVs using rapidly degrading synthetic scaffolds and vascular-derived cells, observing rapid host cell infiltration and remodeling. However, valve coaptation declined over time, leading to mild-to-moderate regurgitation, highlighting the need for improved stent and valve designs [31].

In recent years, ADSCs have attracted attention for their regenerative potential in TEHVs. They enhance tissue integration through paracrine signaling, ECM remodeling, and differentiation into relevant cell types [32]. ADSCs possess strong immunomodulatory and anti-inflammatory properties, releasing growth factors and cytokines such as VEGF, TGF-β, and IL-10, which promote angiogenesis, reduce apoptosis, and mitigate fibrosis, enhancing tissue integration and healing. Moreover, ADSCs inhibit pro-inflammatory immune responses by suppressing the activity of T cells, dendritic cells, and macrophages, while promoting anti-inflammatory cytokine production. This immune regulation reduces inflammation and supports graft acceptance [33].

In our team’s initial attempt, we seeded porcine acellular scaffolds with freshly isolated ADSCs, without prior differentiation or bioreactor conditioning. Harpa et al. found that while acellular xenogeneic valves were stable, non-immunogenic, non-thrombogenic, and non-calcifying, with excellent hemodynamic performance, ADSC-seeding did not enhance regeneration, with signs of right ventricular failure and progressive regurgitation emerging one-month post-implantation. Explanted scaffolds showed host tissue covering the leaflets but lacked cellular infiltration, as most seeded cells died within days, indicating their vulnerability to dynamic pressure and flow [34, 35]. Subsequent in vitro studies using bioreactors confirmed significant cell loss under dynamic conditions in seeded scaffolds, emphasizing the need for improved seeding and conditioning techniques to enhance scaffold integration and stem cell survival [36].

4.1 Impact of ADSC-seeding on postoperative complications and survival outcomes

This study represents our team's second attempt at developing living heart valves using ovine allografts. We refined recellularization techniques by differentiating ADSCs into FBs and ECs-like cells, followed by static and dynamic seeding and bioreactor conditioning before orthotopic implantation in pulmonary position in sheep. While previous studies have focused on mechanical properties, biocompatibility, integration, and immunogenicity, our study addresses the gap in understanding age-dependent postoperative complications, survival outcomes, and CPB-related effects in large animal models. We hypothesized that clinical observations, such as fewer postoperative complications, improved recovery and survival might provide indirect evidence of tissue integration and healing. In vitro analysis of the mechanical properties of the valves and histological examination of the explants were beyond the scope of this study, as they were addressed by Movileanu et al. [23], part of the same research project. No significant differences in postoperative complications were observed between the DECELL and CELL groups, suggesting that ADSC-seeding did not influence complication rates. However, the CELL group showed a trend toward reduced thoracic drainage (Table 1), and an absence of pleural or pericardial effusions, ascites, and endocarditis (Table 3), likely due to the immunomodulatory and anti-inflammatory properties of ADSCs. On the other hand, findings from Movileanu et al., demonstrated that acellular pulmonary valves in juvenile sheep provided excellent hemodynamics, lacked immunogenic and inflammatory responses, were non-thrombogenic, did not calcify, and provided an effective substrate for cell repopulation. They found that ADSC-seeded valves maintained comparable hemodynamic performance to acellular valves while showing enhanced cellular integration. In the post-implantation phase, valve functionality was serially assessed via echocardiography, confirming normal cusps mobility, coaptation, and valve competence, with no signs of structural failure. Additionally, gross inspection at explantation revealed intact valve leaflets without fibrotic thickening, supporting preserved mechanical integrity. Pre-implantation, TEHVs underwent a preconditioning process in a bioreactor, which gradually increased flow rate, pressure, and frequency to simulate physiological pulmonary circulation. This process allowed us to evaluate several key mechanical properties: (1) Valve opening dynamics: TEHVs demonstrated wide opening with a mean geometrical orifice area of ~ 1.4 cm2 for valves with a mean external diameter of ~ 16 mm, confirming proper cusps mobility and flexibility; (2) Closure integrity: The seeded valves achieved perfect central closure with full cusps coaptation, indicating structural stability under pulsatile flow conditions; (3) Tissue resilience: Throughout conditioning, the valves withstood progressive hemodynamic loading without signs of tears, lacerations, or mechanical failure, supporting their durability under physiological stress [23]. Moreover, they noted that ADSC-seeded valves retained leaflet size, suppleness, and functionality for six months post-implantation, with α-smooth muscle actin-positive cells predominantly in the sinus and fibrosa of the leaflets, and endothelial cells covering most surfaces. H&E staining confirmed cellular infiltration, while α-SMA immunohistochemistry showed similar myofibroblast infiltration in ADSC-seeded and DECELL-valves (Base: ~ 40 vs. ~ 35 cells/HPF [cells per high power field]; Mid-leaflet: ~ 30 vs. ~ 25 cells/HPF; Tip: ~ 20 vs. ~ 18 cells/HPF) [23]. The availability of sheep-specific antibodies limited some immunohistochemical assessments, but future research will incorporate broader molecular analyses. Quantitative mechanical testing (e.g., tensile strength, elasticity, and burst pressure) would provide further insights into scaffold durability. In future studies, we plan to perform ex vivo mechanical testing of explanted TEHVs to assess post-implantation structural integrity, to expand bioreactor conditioning studies to include real-time pressure-tracking and force measurements, further validating valve resilience before implantation, and to integrate computational fluid dynamics (CFD) modeling to correlate hemodynamic forces with valve remodeling over time. A larger sample size and extended follow-up, including assessment of inflammatory markers such as IL-6 and TNF-alpha, are needed to better evaluate the role of ADSCs in reducing inflammation, promoting tissue remodeling, and exerting immunomodulatory effects. Additionally, cell tracking techniques would be beneficial to determine whether infiltrating cells originate from the initially seeded ADSCs or are host-derived. These additional analyses will help establish mechanical benchmarks for TEHVs performance and optimize scaffold designs for clinical translation. Systemic inflammation has also been reported as a key factor influencing clinical outcomes in other cardiac pathologies, such as undifferentiated pleomorphic cardiac sarcoma, where elevated inflammatory markers correlated directly with disease activity and prognosis [37].

Our team's results, as previously presented by Harpa et al. and Movileanu et al., highlight the essential role of differentiation, proper seeding, and bioreactor preconditioning in maintaining cell viability and functionality in dynamic environments such as pulmonary circulation [23, 34, 35]. In Movileanu et al. we showed that ADSCs were successfully differentiated into ECs- And FBs-like cells, confirmed by immunofluorescence microscopy for endothelial (CD31, eNOS, von Willebrand factor) and fibroblast (vimentin, Pro-4-hydroxylase, collagen type I) markers. Semi-quantitative analysis showed that ~ 68% of EC-differentiated and ~ 75% of FB-differentiated ADSCs expressed key lineage markers, with a significant marker upregulation in differentiated vs. undifferentiated cells. H&E staining of seeded and preconditioned TEHVs (not implanted) confirmed ECs surface coverage and the presence of FBs at the leaflets base and within the adventitia. These findings confirmed the feasibility of ADSCs differentiation and their regenerative potential [23]. Steinhoff et al. found that static reseeding with autologous myofibroblasts and ECs showed normal valve function up to 3 months in a sheep model of orthotopic pulmonary valve implantation, but histological signs of inflammatory reactions to subvalvar muscle leading to calcifications, were observed also [38]. These findings underscore the importance of dynamic seeding and preconditioning in a bioreactor, as employed in our study, to improve integration and reduce adverse reactions. Also, the use of differentiated FBs and ECs-like in our study aligns with Tudorache et al. [29], where mature endothelial cells demonstrated superior performance compared to incompletely differentiated or undifferentiated cells in studies like Theodoridis et al. [30] and Harpa et al. [34]. The Driessen-Mol study showed that scaffold flexibility and biodegradability influence repopulation and durability [31]. Combined with the anti-inflammatory effects of ADSCs, our results may reflect an interplay between scaffold design and the immunomodulatory properties of the seeded cells, resulting in less postoperative complications and improved survival in CELL group.

Although the six-month survival rate was 57.9%, with no significant differences between the DECELL and CELL groups, the CELL group showed a trend toward higher survival (60% vs. 48.2%) (Fig. 9C). The combined pro-angiogenic, anti-fibrotic and immunomodulatory effects of ADSCs improve vascularization and tissue integration, explaining the observed trend toward better clinical outcomes. The lack of statistical significance in survival rates between valve types (p = 0.7768) may be attributed to the fact that both valve types were based on the same decellularized scaffold. The small sample size and relatively short follow-up might not have been sufficient to reveal long-term survival benefits associated with ADSC-seeded valves. Exploring this trend in larger cohorts and a longer follow-up could provide more robust evidence to confirm these findings.

4.2 Age-specific surgical outcomes

Most studies on TEPVs in humans and animals have focused on single age groups, offering limited insights into age-related differences [39, 40]. Our study revealed significantly longer operative times in adult sheep (Fig. 5A, B, C), primarily due to tissue fragility and technical challenges, with aortic wall rupture prolonging procedures by up to 30%. In one case, it resulted in cardiopulmonary arrest, hemorrhagic shock, and intraoperative death, while in two cases, surgical repair was successful without further complications. Age-related changes, such as increased collagen cross-linking, fragmentation and calcification of elastin fibers, contribute to reduced vascular elasticity and increased tissue stiffness [41, 42]. Additionally, aging increases fibrotic tissue formation, altering the biomechanical properties of tissues and making them more prone to mechanical stress and injury, which complicates surgical manipulation in adult sheep, as previously reported [43]. Despite these challenges, our operative times were comparable with those reported by Knirsch et al., who reported CPB times of 65–75 min and total operative times exceeding 200 min [44]. Vis et al. reported longer CPB times (163 min vs. 75 min in our study), likely due to their more complex on-pump on cardiac arrest procedures [40]. Age-based analysis showed no significant differences between groups; however, juvenile sheep experienced more frequent early and late complications (Tables 5 and 6) and reduced survival (Fig. 9B), though not statistically significant. This trend reflects heightened inflammatory responses and poorer outcomes, aligning with pediatric cardiac surgery findings, where younger patients face higher rates of complications such as infections and effusions [45]. Although juveniles may initially appear better suited for these surgeries, the long-term effects of CPB and related stressors contribute to increased late-stage morbidity and mortality. In contrast, adult sheep experienced fewer late-stage complications despite more challenging intraoperative conditions, demonstrating greater resilience to the chronic effects of surgery and CPB, ultimately leading to higher survival rates, a trend also noted in human studies [45].

Although exact data are limited, the perioperative and short-term mortality in sheep undergoing heart surgeries under CPB range from 10 to 33% for the perioperative period (within 48 h) and from 17 to 50% for the first 30 days postoperatively [46]. However, data on long-term survival remain ambiguous, with limited time-specific survival rates. Our study's 30-day survival rate (84.2%) (Fig. 9A) aligns with that reported by Katz et al. (84%) in a similar study [19]. Minimally invasive approaches, such as percutaneous pulmonary valve implantation have shown improved outcomes due to elimination of CPB, reduced inflammatory responses and surgical trauma, leading to faster recovery and improved survival rates. Attmann et al. and Kim et al. reported survival rates of 66.7% over three months in juvenile sheep and a 33.3% mortality rate over six months in adult sheep, respectively, following transcatheter pulmonary valve implantation using self-expanding nitinol valved stents [47, 48]. Our study showed a comparable survival rate of 63.2%. Age-specific surgical strategies and tailored CPB protocols can improve surgical outcomes and survival rates, even without advanced technologies that eliminate the need for CPB. Nevertheless, comparative future studies evaluating conventional CPB-assisted procedures and percutaneous techniques could further optimize surgical protocols and improve long-term outcomes.

4.3 Effects of cardiopulmonary bypass on ovine homeostasis

While the metabolic effects of CPB, such as hyperlactatemia and anemia, are well-documented in humans [3, 49], comparable studies in sheep are scarce. Previous research has focused on specific aspects, such as fetal CPB strategies [50] and baseline physiological changes during sham valve surgeries [51], but age-related CPB effects remain understudied. Our study addresses this gap by providing a comprehensive analysis of age-specific surgical outcomes and CPB-related physiological effects.

CPB leads to physiological changes in blood gas levels and electrolyte imbalances, leading to systemic inflammatory responses and metabolic changes [21]. Human studies have associated elevated lactate levels due to inadequate oxygen delivery and reperfusion injury, with enhanced inflammatory responses, all contributing to unfavorable postoperative outcomes in both adult and pediatric patients [3, 52,53,54,55]. Ranucci et al. identified hyperlactatemia during CPB and central venous oxygen saturation (ScVO2) < 68% as predictors of increased morbidity and mortality [56]. In our study, prolonged CPB in adult sheep led to significantly higher lactate levels (Fig. 6A, B), metabolic acidosis, and transient anemia, suggesting heightened systemic inflammatory responses Previous studies have also associated prolonged CPB with the accumulation of inflammatory mediators, such as cytokines and acute-phase proteins, exacerbating tissue damage and impairing healing processes [3, 56].

Faustich et al. reported significant decreases in hemoglobin, red blood cell count, hematocrit and albumin post-CPB, attributing these changes to a combination of blood loss, CPB-induced hemodilution, and surgical stress [51], findings consistent with our results. Lower hemoglobin and hematocrit levels in adult sheep during CPB (Fig. 6C, D) highlight their greater susceptibility to CPB-induced anemia, a common complication in cardiac surgery [3]. Hemodilution caused by the priming solution and red blood cell destruction due to non-physiological flow in the ECC circuit are major contributors to anemia in CPB adult models [2, 57]. In contrast, juvenile sheep exhibited a stronger hematopoietic response and better adaptation to CPB stress. Over the 6-month follow-up, three deaths in juvenile sheep with decellularized valves were attributed to endocarditis, likely due to the heightened inflammatory response following CPB and increased susceptibility to bacterial adhesion, as previously reported [22]. Although ADSC-seeding did not significantly reduce acute surgical risks, our observations emphasize the need for strategies to mitigate late-stage inflammatory complications, aligning with findings from other small animal and human studies [2, 3].

4.4 Coagulation homeostasis

This study provides insights into the age-specific anticoagulation management, focusing on heparin dosing, pre- And post-CPB ACT values, and protamine doses. Thrombosis of the ECC circuit can be fatal for patients, while inadequate control may contribute to postoperative hemorrhage, a major cause of morbidity and mortality in cardiac surgery [58]. ACT was monitored intraoperatively every 30 min to assess the adequacy of the heparin regimen, with reference values between 80 and 120 s [59].

Juvenile sheep required significantly higher intravenous heparin doses (Fig. 8A, B) to achieve adequate anticoagulation, likely due to faster metabolism and clearance rates, while ECC-administered doses remained similar across age groups, bypassing metabolic variability. The significant difference in pre-CPB ACT (Fig. 8C) suggest age-specific baseline coagulation profiles, with juveniles requiring more time to reach target ACT levels, as previously reported [2, 3]. The heparin-to-protamine ratio was effectively managed in both groups, maintaining adequate coagulation without excessive bleeding or thrombotic events. Despite initial pre-CPB ACT differences, post-CPB ACT values were comparable (Fig. 8D), indicating similar coagulation normalization and effective heparin reversal. The greater variability in post-CPB ACT in juveniles, reflected by a higher standard deviation, may be attributed to age-related physiological differences or individualized responses to protamine, as described in previous studies [2, 60, 61].

Unlike previous studies that focused on single age cohorts [29,30,31, 34, 35, 38, 40], our study demonstrates clear age-specific differences in surgical outcomes and anticoagulation management, emphasizing the distinct effects of CPB on juvenile and adult sheep. To the best of our knowledge, this is the first study to comprehensively analyze these physiological and hematological differences during heart surgeries. The observed variability between age groups underscores the need for tailored CPB and coagulation protocols to optimize outcomes and address age-related challenges.

4.5 Key findings and clinical implications

We have summarized the key findings and the broader implications of this study in Table 10. Ultimately, achieving a truly living heart valve that fulfils all performance criteria—including hemodynamic functionality, durability, and the ability to grow and self-repair—while also being readily available off-the-shelf, requires further research. This includes identifying optimal stem cell sources, refining recellularization techniques, and developing efficient seeding methods to enhance scaffold integration and regeneration. The development of more standardized seeding and conditioning protocols is necessary to improve batch-to-batch reproducibility, a crucial factor for eventual clinical translation and regulatory approval. Continued advancements in this field are essential to bridge the gap between the two approaches—acellular vs. seeded scaffolds—in order to achieve the next generation of TEHVs. Comparative studies between cell-seeded and cell-free scaffolds will help clarify whether endogenous repopulation alone is sufficient for long-term valve function, a question of significant clinical importance given that cell-free approaches have gained traction in regenerative medicine. Cell-tracking strategies would be beneficial to determine whether implanted ADSCs contribute to tissue remodeling or if host-derived cells primarily drive neotissue formation. Larger preclinical studies and long-term functional validation—extending follow-up beyond six months to assess chronic inflammation, neotissue formation, and valve function—along with careful consideration of manufacturing and regulatory pathways, will be essential for progressing toward clinical trials and market translation. By addressing these translational challenges, TEPVs could eventually provide an off-the-shelf solution for congenital and acquired pulmonary valve disease, improving outcomes for pediatric and adult patients alike.

Table 10 Key findings and the broader implications of this study4.6 Study limitations

Several limitations of this study were identified: (1) The small sample size may have been insufficient to detect subtle differences in postoperative complications or mortality, particularly regarding the effects of ADSC-seeding. To adhere to ethical principles and the 3R framework (replacement, reduction, and refinement), the number of animals was deliberately minimized; (2) Age distribution: The inclusion of both juvenile and adult sheep introduced variability, potentially complicating comparisons between the DECELL and CELL groups. A more homogeneous age group could have reduced this variability, providing clearer insights into the specific effects of ADSC-seeding; (3) Follow-up period: The study focused on short- and medium-term surgical outcomes and physiological changes associated with CPB, with a follow-up period limited to six months. A longer observation period would provide valuable insights into chronic complications, and the long-term effects of ADSC-seeded valves on tissue integration and regeneration; (4) Detailed in vitro analysis including mechanical testing (tensile strength, elasticity, burst pressure) and expanded bioreactor-based assessments, are planned for future studies, to further validate TEPVs structural integrity and long-term performance. In vitro differentiation assays of TEHVs pre-implantation and histological examination of the explanted valves were beyond the scope of this study; therefore, the mechanisms of ADSC-mediated tissue integration were not directly assessed. These aspects will be addressed in upcoming research; (5) The absence of quantified inflammatory markers, such as IL-6 and TNF-alpha would provide a more precise assessment of the inflammatory response. Future studies will incorporate comprehensive inflammatory profiling to better elucidate the role of ADSCs in modulating immune responses and inflammation.

In conclusion, the following can be affirmed:

Our study demonstrates that age significantly impacts surgical complexity and outcomes. Adult sheep, despite greater intraoperative challenges, demonstrated better long-term survival, making them more suitable for chronic experimental studies. Juvenile sheep, while undergoing less demanding procedures, experienced more late-stage complications, indicating their suitability for short-term studies in conventional CPB-assisted procedures.

Cardiopulmonary bypass significantly affected metabolic markers, with adult sheep showing a greater tendency for metabolic acidosis and anemia, reflecting increased metabolic stress. The higher mortality rate in juvenile, suggests an increased inflammatory response post-CPB, emphasizing the need for age-specific surgical strategies and CPB protocols.

ADSC-seeding did not significantly impact operative parameters, complications or survival rates. Further research with an extended follow-up period is needed to better understand the long-term effects of ADSC-seeding on tissue integration and regeneration.

Our findings highlight the importance of age-specific anticoagulation protocols for optimizing perioperative management and preventing complications. While CPB-related coagulopathy was effectively managed across age groups, individualized anticoagulation strategies for juveniles could further minimize variability in postoperative coagulation responses.

Therefore, valve type alone cannot be sufficient to address the complexities of postoperative recovery, particularly in the context of age-related variability and CPB-induced metabolic stress.