Every year, thousands of aortic valve replacements must take place due to valve diseases. Tissue-engineered heart valves represent promising valve substitutes with remodeling, regeneration, and growth capabilities. However, the accurate reproduction of the complex three-dimensional (3D) anatomy of the aortic valve remains a challenge for current biofabrication methods. We present a novel technique for rapid fabrication of native-like tricuspid aortic valve scaffolds made of an alginate-based hydrogel. Using this technique, a sodium alginate hydrogel formulation is injected into a mold produced using a custom-made sugar glass 3D printer. The mold is then dissolved using a custom-made dissolving module, revealing the aortic valve scaffold. To assess the reproducibility of the technique, three scaffolds were thoroughly compared. CT (computed tomography) scans showed that the scaffolds respect the complex native geometry with minimal variations. The scaffolds were then tested in a cardiac bioreactor specially designed to reproduce physiological flow and pressure (aortic and ventricular) conditions. The flow and pressure profiles were similar to the physiological ones for the three valve scaffolds, with small variabilities. These early results establish the functional repeatability of this new biofabrication method and suggest its application for rapid fabrication of ready-to-use cell-seeded sodium alginate scaffolds for heart valve tissue engineering.

The fabrication of anatomically accurate, cellularized heart valve substitutes remains a significant challenge in tissue engineering, particularly for pediatric and patient-specific applications. While three-dimensional (3D) bioprinting enables the creation of complex geometries, it often compromises cell viability and lacks the precision required for small-scale constructs. In this study, we present a high-fidelity, reproducible molding technique using 3D-printed sugar glass molds to engineer custom, alginate-based hydrogel cellularized heart valves. Human adipose-derived stromal cells (ASCs) were used as the cell source due to their accessibility and regenerative potential. This approach overcomes the limitations of conventional molding and bioprinting by enabling the reproduction of intricate anatomical features, including the sinuses of Valsalva, which are critical for physiological hemodynamics. The molding method maintains high cell viability (>90%) at the time of fabrication and the process supports both scalability and automation. Sugar glass molds for valve sizes from 16 to 26 mm inner diameter were printed with 90% of the mold surface within a ±0.3 mm deviation of the reference computer-aided design model. Cellularized valves cultured in a custom perfusion bioreactor retained structural integrity and cell viability over a 14 d period. This biofabrication strategy offers a promising platform for engineering patient-specific heart valves and also lays the groundwork for in vitro disease modeling, including valve mineralization, using living cells such as ASCs.

Current pediatric heart valve protheses lack growth potential, leading to repeated surgeries and long-term complications. Tissue-engineered heart valves (TEHVs), composed of living tissue, offer the ability to remodel and may accommodate somatic growth. This study introduces a sutureless fabrication strategy for TEHV designed to eliminate suture-related weak points and preserve valve geometry during in vitro maturation through a novel evolutive support system. This system was used to form and maintain the geometry of a fibrin-based valve scaffold embedded with human adipose-derived stromal cells during in vitro culture by mechanically constraining tissue contraction in all directions except thickness. Dimensional analysis confirmed geometry preservation throughout 28 days of perfusion bioreactor culture. At the end of the culture period, the matured valve was released from the evolution support system, after which the valve contracted to stable dimensions of approximately 16 mm in diameter, 23 mm in height, and 400 µm in thickness, corresponding to reductions of 26%, 43%, and 84% in diameter, height, and thickness, respectively, relative to the initial molded geometry. These ratios provide critical design guidance for pediatric target dimensions. Cell viability exceeded 90% upon molding, and cell density increased nearly 100-fold during culture. Histology revealed progressive collagen deposition, which correlated with improved mechanical properties. Stiffness increased nearly 9-fold and puncture strength 2.5-fold from fabrication to day 28. This reproducible sutureless strategy enables geometry retention of TEHV by guiding tissue contraction during in vitro culture, paving the way for pulsatile conditioning and further maturation to achieve mechanical properties approaching those of native valves and ensure long-term functionality.

Urethral reconstruction is required for a variety of congenital and acquired conditions. Current grafting techniques rely on autologous tissues such as buccal mucosa, which are often limited by donor-site morbidity. Biomaterials could be an alternative. However, they may present immunological risks and inconsistent surgical outcomes. The tissue-engineering self-assembly approach, which uses autologous patient cells to generate tissue constructs without exogenous biomaterials, offers a promising alternative by improving graft integration. Such natural tissue-engineered constructs exhibit promising histological similarity to native urethral tissue and hold potential for clinical application. However, graft selection for surgery remains subjective, lacking standardized mechanical criteria to ensure consistent quality. Previous efforts to quantify graft properties through uniaxial tensile testing have proven informative but require cutting the tissue into standardized specimens and are therefore not readily suited to routine quality-control workflows. To address this, we investigated puncture testing as a simpler practical alternative that does not require tissue cutting and assessed its correlation with tensile testing and surgeon-based evaluation. Self-assembled dermal and urethral constructs were evaluated using uniaxial tensile and puncture tests, and results were correlated with the surgeon’s qualitative assessments during graft handling. Our findings reveal a strong linear relationship between two mechanical testing techniques. Puncture testing also showed a strong relationship with surgeon-based qualitative assessments of graft handling. This study highlights the potential of puncture testing as a practical mechanical assessment tool for characterizing graft mechanical behavior in the context of surgical handling, and offers a pathway toward the development of standardized, objective quality control protocols for tissue-engineered urethral grafts prior to implantation.