Three-dimensional printed biomimetic multilayer scaffolds coordinated with sleep-related small extracellular vesicles: A strategy for extracellular matrix homeostasis and macrophage polarization to enhance osteochondral regeneration

Cartilage defects resulting from injury or degeneration are a common clinical problem, and due to its avascular nature, articular cartilage has poor self-healing capacity. Three-dimensional (3D) bioprinting has attracted great attention in tissue engineering. Melatonin (MT), a hormone mainly secreted at night, plays an important role in tissue repair. Small extracellular vesicles (sEV) are considered ideal drug delivery vehicles and MT-sEV (sleep-related sEV) have the potential ability to promote cartilage regeneration. Here, biomimetic multilayer scaffolds were fabricated using 3D bioprinting. A double network hydrogel, composed of methacrylated hyaluronic acid and gelatin methacryloyl (HG), was prepared. MT-sEV and HG hydrogel were used to create a cartilage layer. A bone layer was formed using poly(epsilon-caprolactone) and hydroxyapatite ultralong nanowires. Additionally, two bioinks were alternately printed at the interface layer. The results of RNA sequencing revealed the potential regulatory mechanisms. MT-sEV showed promotional effects on cell migration, proliferation, chondrogenic differentiation, and extracellular matrix (ECM) deposition. Moreover, MT-sEV altered macrophage polarization and regulated the expression of inflammatory cytokines. In vivo experiments demonstrated that the biomimetic multilayer scaffolds promoted cartilage regeneration. These experiments demonstrated the ability of MT-sEV to regulate the immune microenvironment and promote the secretion of ECM, providing a promising strategy for cartilage regeneration. Our research revealed MT-sEV's bioactive functions for extracellular matrix homeostasis and macrophage polarization. And we utilized 3D bioprinting technology to construct a multilayer, biomimetic scaffold incorporated with MT-sEV to promote enhanced osteochondral regeneration.image