Stage-specific MSC spheroid fusion enables bottom-up formation of stage-defined hierarchical cartilage graft.

Engineering articular cartilage with hierarchical architecture and effective interfacial integration remains a major challenge. Although bottom-up spheroid assembly offers a promising biofabrication strategy, how chondrogenic maturation influences spheroid fusion and boundary remodeling remains incompletely defined. Here, we investigated differentiation stage as a programmable determinant of the phenotype and assembly behavior of mesenchymal stem cell (MSC)-derived chondrogenic spheroids. Exploratory RNA-seq pathway mapping, complemented by biologically replicated RT-qPCR, indicated stage- and cell-source-associated differences in cartilage matrix and maturation-related programs in bone marrow-derived MSCs (BM-MSCs) and umbilical cord-derived MSCs (UC-MSCs). Early-stage spheroids exhibited greater fusion capacity than mature spheroids, whereas incorporation of undifferentiated MSCs promoted the integration of mature chondrogenic spheroids and reduced persistent inter-spheroid boundaries. Sequential assembly of Day-3, Day-7, and Day-14 spheroids generated hierarchical constructs with stage-associated regional differences in cartilage extracellular matrix deposition. Incorporating an undifferentiated MSC layer at the bone-facing interface further supported structural continuity with a porous bone substitute in vitro. In an exploratory porcine pilot study involving two animals, implantation of MSC-derived spheroid grafts into cartilage defects was technically feasible. At 6 months, the repair tissue exhibited variable proteoglycan staining and heterogeneous collagen type II immunoreactivity, while selected sections showed tissue apposition at the graft-host interface. The limited animal-level sample size precluded conclusions regarding repair efficacy, cartilage quality, or superiority over autologous cartilage implantation. Collectively, these findings identify differentiation stage as an intrinsic bioassembly variable and establish a stage-programmed strategy for constructing hierarchical cartilage grafts. Both BM-MSCs and UC-MSCs supported stage-defined assembly in vitro, warranting further evaluation of UC-MSCs as a potential allogeneic cell source in adequately powered studies.