The clinical translation of mesenchymal stem cell (MSC)-based therapies for cartilage repair is limited by inefficient lineage specification and the frequent formation of mechanically inferior fibrocartilage. Here, we present a gene-activated scaffold strategy that couples transient mRNA delivery with a structurally defined biomaterial to program ectopic chondrogenesis. Lipid nanoparticle-mediated delivery of SOX9 mRNA within a microfluidically engineered porous scaffold induces a transcriptional response in MSCs, initiating a chondrogenic program without the need for sustained growth factor stimulation. In three-dimensional spheroid and scaffold models, SOX9 mRNA drives robust upregulation of cartilage-specific genes, including ACAN and COL2A1, while limiting fibrocartilage- and hypertrophy-associated markers. Transcriptomic analysis reveals that this response is characterized by coordinated activation of extracellular matrix organization and regulatory signaling pathways, consistent with the establishment of a self-reinforcing differentiation trajectory. Importantly, this transcriptional program is preserved within the scaffold microenvironment, enabling efficient ectopic lineage specification. Following subcutaneous implantation, MSC-laden scaffolds exhibit progressive extracellular matrix deposition, sustained expression of chondrogenic markers, and the formation of lacunae-like structures over time, indicative of tissue maturation. While this ectopic model does not recapitulate the native joint environment, the persistence of cartilage-like features supports the durability of the initial transcriptional programming. Together, these findings demonstrate that a transient mRNA-encoded transcriptional cue, when combined with a permissive three-dimensional scaffold, is sufficient to initiate and sustain chondrogenesis across biological scales. This work establishes a framework for mRNA-enabled biomaterials to direct cell fate and provides a foundation for the development of single-stage strategies for cartilage regeneration.
