Enzymatically\nCross-Linked Silk Fibroin-Based Hierarchical Scaffolds for Osteochondral\nRegeneration

Abstract

Osteochondral\n(OC) regeneration faces several limitations in orthopedic surgery,\nowing to the complexity of the OC tissue that simultaneously entails\nthe restoration of articular cartilage and subchondral bone diseases.\nIn this study, novel biofunctional hierarchical scaffolds composed\nof a horseradish peroxidase (HRP)-cross-linked silk fibroin (SF) cartilage-like\nlayer (HRP-SF layer) fully integrated into a HRP-SF/ZnSr-doped β-tricalcium\nphosphate (β-TCP) subchondral bone-like layer (HRP-SF/dTCP layer)\nwere proposed as a promising strategy for OC tissue regeneration.\nFor comparative purposes, a similar bilayered structure produced with\nno ion incorporation (HRP-SF/TCP layer) was used. A homogeneous porosity\ndistribution was achieved throughout the scaffolds, as shown by micro-computed\ntomography analysis. The ion-doped bilayered scaffolds presented a\nwet compressive modulus (226.56 ± 60.34 kPa) and dynamic mechanical\nproperties (ranging from 403.56 ± 111.62 to 593.56 ± 206.90\nkPa) superior to that of the control bilayered scaffolds (189.18 ±\n90.80 kPa and ranging from 262.72 ± 59.92 to 347.68 ± 93.37\nkPa, respectively). Apatite crystal formation, after immersion in\nsimulated body fluid (SBF), was observed in the subchondral bone-like\nlayers for the scaffolds incorporating TCP powders. Human osteoblasts\n(hOBs) and human articular chondrocytes (hACs) were co-cultured onto\nthe bilayered structures and monocultured in the respective cartilage\nand subchondral bone half of the partitioned scaffolds. Both cell\ntypes showed good adhesion and proliferation in the scaffold compartments,\nas well as adequate integration of the interface regions. Osteoblasts\nproduced a mineralized extracellular matrix (ECM) in the subchondral\nbone-like layers, and chondrocytes showed GAG deposition. The gene\nexpression profile was different in the distinct zones of the bilayered\nconstructs, and the intermediate regions showed pre-hypertrophic chondrocyte\ngene expression, especially on the BdTCP constructs. Immunofluorescence\nanalysis supported these observations. This study showed that the\nproposed bilayered scaffolds allowed a specific stimulation of the\nchondrogenic and osteogenic cells in the co-culture system together\nwith the formation of an osteochondral-like tissue interface. Hence,\nthe structural adaptability, suitable mechanical properties, and biological\nperformance of the hierarchical scaffolds make these constructs a\ndesired strategy for OC defect regeneration.