Mechano-Hypoxia Programs a Coordinated Chondrogenic–Osteochondral Transcriptome in Co-Cultures of Human Nasal Chondrocytes and Bone Marrow–Derived Mesenchymal Stromal Cells

Nasal chondrocyte–bone marrow mesenchymal stromal/stem cell (NC-BMSC) co-culture can enhance cartilage matrix formation, but whether this response remains separated from hypertrophic or osteochondral maturation under combined hypoxic and mechanical stimulation is unclear. Here, human NCs and BMSCs were seeded onto type I collagen scaffolds as monocultures or 1:3 NC:BMSC co-cultures, cultured for 6 weeks under hypoxia in chondrogenic medium, and then subjected to 7 days of intermittent dynamic compression. Construct phenotype was assessed using histology, collagen immunofluorescence, glycosaminoglycan and DNA quantification, unconfined compression testing, RT-qPCR, and endpoint bulk RNA sequencing. Co-culture constructs retained strong Safranin O-positive matrix and collagen II staining, whereas BMSC monocultures showed loss of proteoglycan-rich matrix by the endpoint. Measured glycosaminoglycan content in co-culture significantly exceeded the weighted expected value from the corresponding monocultures, indicating chondro-induction beyond additive cell-source contributions. Co-culture also produced significantly higher equilibrium and peak compressive moduli than donor-matched NC monocultures. RT-qPCR showed increased ACAN and SOX9 expression in co-culture, but also increased COL10A1, indicating that hypertrophic features were not fully suppressed. Endpoint RNA sequencing separated co-culture from NC monoculture and identified overlapping proliferation-, extracellular matrix-, and differentiation-associated programs. Co-culture upregulated proteoglycan- and cartilage-associated genes together with mineralization-, angiogenesis-, and remodeling-associated genes including SPP1, IBSP, DMP1, ALPL, and VEGFA. Mechanotransduction-associated genes and immediate-early transcriptional regulators were also enriched under the applied loading regimen. These findings show that NC-BMSC co-culture under hypoxic dynamic compression enhances cartilage-like matrix formation and mechanical function, but the improved tissue phenotype remains coupled to an osteochondral remodeling transcriptome. Future optimization should focus on uncoupling matrix induction from hypertrophic and mineralization-associated maturation.