Dual crosslinkable hybrid alginate/ methacrylated gelatin/ cartilage extracellular matrix hydrogels for cartilage printing

Abstract

A novel tri-component bioink composed of methacrylated gelatin (GelMA), alginate, and decellularized cartilage extracellular matrix (dcECM) was developed to enhance both the printability and biological performance of hydrogel scaffolds for cartilage tissue engineering. The incorporation of cartilage-specific dcECM was employed as a key strategy to provide a biologically instructive microenvironment while maintaining suitable rheological properties for extrusion-based bioprinting. Rheological analyses revealed pronounced shear-thinning behavior, good thermal stability, and a predominantly elastic response, which collectively enabled smooth extrusion during 3D printing. Mechanical characterization revealed that increasing GelMA content significantly enhanced compressive modulus and strength via covalent crosslinking, whereas dcECM incorporation led to a moderate reduction in stiffness, a trade-off accepted for improved biological functionality. Biological assessments confirmed that the alginate/GelMA/dcECM scaffolds supported chondrocyte adhesion, proliferation, and viability, while facilitating native extracellular matrix deposition. Histological analyses showed enhanced glycosaminoglycan accumulation and more homogeneous cellular distribution in scaffolds containing higher dcECM content. Importantly, the optimized cell-laden bioprinted formulation exhibited homogeneous cell distribution and preserved construct integrity after 14 days of culture, as confirmed by DAPI nuclear staining. To evaluate preliminary in vivo biocompatibility, cell-free 3D-printed alginate/GelMA/dcECM scaffolds were implanted subcutaneously in Wistar rats for 28 days. No systemic adverse effects or macroscopic signs of infection were observed. Histological examination (H&E and Masson's Trichrome) indicated normal tissue architecture, mild localized inflammatory response, and complete scaffold degradation, confirming non-toxic resorption. Overall, these findings underscore the synergistic potential of natural polymers and dcECM in creating a bioprintable, biologically instructive, and mechanically tunable platform for cartilage regeneration.