BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration. PURPOSE: To tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD. STUDY DESIGN: An extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD. METHODS: An optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production. RESULTS: Among all cocultured groups, hAF cells and hMSCs in the ratio of 2: 1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers. CONCLUSIONS: We demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The