Development of Intervertebral Disc Organoids through Directed Differentiation of Mesenchymal Stem Cells and Hierarchical 3D Printing.

Organoid models of early tissue development have been generated for organs such as the brain, kidney, and intestine. However, the development of intervertebral disc (IVD) organoids has rarely been reported. Here, we have developed a protocol to directly differentiate nucleus pulposus (NP) organoids and annulus fibrosus (AF) organoids from human mesenchymal stem cells (hMSCs) using differentiation media supplemented with folic acid (FA) and connective tissue growth factor in a 3D environment. Our findings suggested that FA could potentially promote NP cell generation through modulation of the PI3K-AKT and TGF-β pathways. Utilizing digital light processing 3D printing techniques, we hierarchically constructed biomimetic IVD scaffolds consisting of customized host-guest and silk fibroin hydrogels for NP and AF organoids. Subsequently, we manually assembled IVD scaffolds, NP organoids and AF organoids into complex IVD organoids (IVDOs) exhibiting a central NP-like region surrounded by concentric AF-like structures. Furthermore, the implantation of IVDOs into the goat lumbar spine after discectomy results in anisotropic reconstruction of the IVD. These findings highlight the successful establishment of an in vitro IVD organoid model for future research of disc degenerative diseases and demonstrate a translational therapeutic approach for IVD repair.