ABSTRACT: For nasal reconstruction and the repair of articular cartilage lesions, autologous nasal septal cartilage (NSC) is a promising cell source for engineering hyaline cartilage. However, the low cell yield and high variability of cartilage quality between patients limit clinical scalability. Human NSC were isolated from tissue by enzymatic digestion, and after two passages, were treated with 1 ng/mL transforming growth factor β1 (TGF-β1), 5 ng/mL fibroblast growth factor 2 (FGF-2), or both factors together. Some cells were collected for molecular analyses before 3 weeks of chondrogenic differentiation, after which pellets were subjected to morphological, mechanical, histological, immunofluorescence, and biochemical analyses. Dual-priming resulted in the highest cumulative population doubling, followed by FGF-2, then TGF-β1. Treatment-dependent differences in cartilage matrix-forming genes largely converged in 3D constructs and showed little correlation, demonstrating the profound influence of culture system on gene expression. FGF-2-treated cells formed larger pellets by area and wet weight, followed by dual-primed cells, with TGF-β1-treated cells forming the smallest pellets with slightly higher Safranin-O staining intensity by area. Between dual-primed and TGF-β1-primed cells, bulk, unbiased RNA sequencing reveals extensive modulation of genes involved in mitotic/chromosome/DNA-maintenance programs, ECM/adhesion/cytoskeletal rewiring, and cell signalling. Principal component analysis demonstrates that, compared to TGF-β1 treatment, dual-priming results in much greater transcriptomic similarity with FGF-2-treated cells. We discuss the interacting regulator effects between both growth factors in driving the phenotype observed under combined treatment. Altogether, our analysis of regulated intracellular signalling pathways and growth factor interactions at the molecular level provides novel, foundational insights into cartilage biology that are essential for advancing tissue engineering strategies and optimizing clinical outcomes.
