Three-dimensional bioprinting combined with natural hydrogels is a promising technology for the treatment of several pathologies and different tissue regeneration. One of the most studied tissues is cartilage, a complex and avascular tissue that displays a limited self-repair capacity after injuries. Herein, the development of alginate-based hydrogels and scaffolds containing different microstructure is presented and the printability of alginate by 3D bioprinting is studied. Rheological characterization was performed for the determination of viscosity and viscoelastic properties of hydrogels and mechanical characterization was carried out for the determination of compressive modulus of alginate hydrogels. All these characteristics were correlated with alginate behaviour during 3D bioprinting process. For the printability evaluation filament diameter, perimeter of the pores, area of the pores and shrinkage of alginate scaffolds were measured. The results demonstrate that alginate microstructure has a great influence on its printability and on hydrogels’ physicochemical properties. Molecular weight of alginate determines its viscosity while M/G ratio determines cross-linking conditions and mechanical properties that vary with cross-linking density. These results suggest the importance of an exhaustive control of the viscoelastic and mechanical properties of alginate hydrogels to obtain structures with high resolution and precision.

This work identifies and describes different material-scaffold geometry combinations for cartilage tissue engineering (CTE). Previously reported potentially interesting scaffold geometries were tuned and printed using bioresorbable polycaprolactone and poly(lactide-b-ethylene) block copolymer. Medical grades of both polymers were 3D printed with fused filament fabrication technology within an ISO 7 classified cleanroom. Resulting scaffolds were then optically, mechanically and biologically tested. Results indicated that a few material-scaffold geometry combinations present potential for excellent cell viability as well as for an enhance of the chondrogenic properties of the cells, hence suggesting their suitability for CTE applications.

Introduction: The clinical translation of biomaterial-based strategies for ear reconstruction remains limited by several challenges, including scaffold design, selection of an optimal cell source, and stability of the new cartilage tissue. Most recent studies still rely on animal-derived cells and fail to demonstrate the long-term maintenance of the chondrogenic phenotype. Methods: In this study, we developed an off-the-shelf poly(lactide-b-ethylene glycol) (PLA-PEG) scaffold combined with clinical-grade alginate (Alg) and cellularized with human nasal chondrocytes for auricular cartilage engineering. Cellularized constructs were evaluated using histological and immunofluorescence analyses to assess extracellular matrix production and quality. In parallel, constructs retrieved after 12 weeks of in vivo implantation were mechanically characterized using a UniVert CellScale testing system to determine variations in compressive strength. Finally, the chondrogenic maturation of the new tissue was evaluated through bulk RNA transcriptomic profiling. Results: Histological, immunofluorescence, and transcriptomic analyses demonstrated robust cartilage matrix deposition with improved compressive properties and high expression of chondrogenic markers, such as ACAN, PRG4, and COL2A1. Furthermore, positive staining for the human-specific Ku80 antibody confirmed the presence of cells of human origin in the newly formed tissue after implantation. Conclusions: Overall, this study provides evidence that the combination of a PLA-PEG + Alg scaffold with human nasal septal chondrocytes represents a promising strategy for future reconstructive applications in auricular tissue engineering.

A novel, user-friendly bioreactor for the cultivation of cellularised membranes for tissue engineering has been successfully designed, manufactured, and validated. This bioreactor features a culture vessel and a cover, the latter equipped with one or more sidewalls to ensure airtightness in two distinct zones, thereby maintaining sterile conditions. The cover, designed to integrate seamlessly with the culture vessel, includes several ports compatible with commercial connectors. This design allows the introduction of cells and culture medium without requiring the opening of the cover, thus preserving sterility. Additionally, the cover is equipped with flanges that effectively press the membrane against the bottom surface of the culture vessel, preventing it from shrinking or shifting. This ensures that cells can properly adhere to the membrane and proliferate. Manufactured under Good Manufacturing Practice (GMP) conditions, the bioreactor supports cultivation in optimal aseptic environments, thereby preventing external contamination. This feature is critical for the safe transportation of cultivated tissue to clinical settings. Validation tests have confirmed the bioreactor’s excellent performance, endorsing its suitability for intended applications in tissue engineering.