Development of Intervertebral Disc Organoids through Directed Differentiation of Mesenchymal Stem Cells and Hierarchical Bioprinting

Abstract 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 fibrosis (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. We found that FA promotes NP generation by regulating the PI3K-AKT and TGF-β pathways. Utilizing digital light processing 3D printing techniques, we hierarchically constructed biomimetic scaffolds consisting of customized host-guest and silk fibroin hydrogels for NP and AF organoids. Subsequently, these were assembled into complex and highly structured IVD organoids (IVDOs) characterized by a central NP-like region surrounded by AF-like structures. Furthermore, the implantation of IVDOs into the goat lumbar spine after IVD discectomy results in anisotropic reconstruction of the IVD and has potential for preventing degenerative disc diseases. These findings highlight the successful development of IVDOs for the study of disc degenerative diseases and underscore the need for advancements toward further clinical translation in treating disc degeneration.