Integrated 3D-bioprinted bilayered gellan gum-based nanocomposite hydrogels with inductive nanoparticles and enhanced pro-angiogenic potential for osteochondral tissue engineering

Osteochondral defects present a significant clinical challenge due to the limited self-healing capacity of cartilage and the complex, graded interface between tissues. This study developed a novel 3D-bioprinted bilayered scaffold designed to mimic the native osteochondral unit. The cartilage-like layer comprised a dual-crosslinked network of gellan gum (GG), oxidized gellan gum (OGG), and decellularized human amniotic membrane (dHAM), reinforced with melatonin-loaded PLGA nanoparticles (MEL@PLGA) to promote chondrogenesis. This layer exhibited a uniform pore size (~442 μm), a compressive modulus of ~172.4 kPa, and controlled degradation over 21 days. Biological evaluations confirmed enhanced glycosaminoglycan (GAG) production and proteoglycan-rich matrix formation, indicating efficient chondrogenic differentiation. The subchondral bone layer consisted of GG, OGG, and silk fibroin (SF), reinforced with strontium-doped montmorillonite (Sr@MMT) nanosilicates (GG-OGG-SF/Sr@MMT). This formulation significantly enhanced mechanical properties (with a compressive modulus up to 466.3 kPa) while simultaneously promoting osteogenesis and angiogenesis. Tensile testing of the bilayered construct revealed a tensile modulus of ~180 kPa confirming strong interfacial integration. In vitro results demonstrated markedly increased alkaline phosphatase (ALP) activity and increased calcium deposition (confirmed by Alizarin Red staining), indicating robust osteogenic differentiation and mineralization. Furthermore, extracts from Sr@MMT-containing scaffolds significantly enhanced HUVEC migration and tube formation, supporting the scaffold's pro-angiogenic potential. The bilayered construct supported region-specific differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), upregulating chondrogenic markers (SOX9, COL2, ACAN) in the cartilage layer and osteogenic markers (RUNX2, COL1, OCN) in the subchondral bone layer. This multifunctional 3D-bioprinted bilayered scaffold offers a promising strategy for osteochondral tissue engineering.