RESEARCH SUMMARY
This study investigated how TGF-β1 and FGF-2, individually and in combination, influence the expansion, molecular state, and subsequent cartilage-forming capacity of human nasoseptal chondrocytes. Chondrocytes from six donors were expanded with TGF-β1, FGF-2, or both growth factors, then formed into 3D microtissue pellets and cultured for 21 days under chondrogenic conditions. Dual TGF-β1/FGF-2 priming produced the greatest cumulative population expansion, while FGF-2-primed cells generated the largest pellets. Despite differences in pellet size and matrix staining, the three priming groups did not differ significantly in equilibrium modulus, total GAG, GAG/DNA, or Bern histological score. RNA sequencing showed that the transcriptomic state of dual-primed cells more closely resembled FGF-2-primed cells than TGF-β1-primed cells, with only 171 differentially expressed genes between the dual and FGF-2 conditions compared with 1246 between the dual and TGF-β1 conditions. FGF-2-containing conditions altered pathways associated with cell cycle regulation, extracellular matrix and adhesion, integrin signaling, PI3K-Akt, Rap1, actin organization, and TGF-β superfamily signaling. TGF-β1/FGF-2 priming also increased expression of BMP2, BMP6, GDF5, and NOG relative to TGF-β1 alone, suggesting coordinated regulation of cartilage maturation pathways. Overall, the study shows that FGF-2 exerts a dominant influence on the molecular phenotype produced by combined TGF-β1/FGF-2 priming, while all tested priming regimens can generate cartilage-like 3D microtissues with broadly comparable biochemical and mechanical properties.
A CellScale MicroTester G2 was used to measure the equilibrium compressive modulus of 3D nasoseptal chondrocyte microtissue pellets after 21 days of chondrogenic culture. Pellets generated from cells previously expanded with TGF-β1, FGF-2, or combined TGF-β1/FGF-2 were placed on a stable platform and compressed with a parallel upper platen. Stepwise stress-relaxation testing was performed at successive tissue strains of 10%, 20%, and 30%. Each strain increment was applied at 2% strain per second and followed by a 300-second hold at constant strain, allowing the pellets to reach mechanical equilibrium. Pellet diameter and platen height were recorded at initial contact, while the MicroTester continuously recorded changes in specimen height and reaction force. Stress was calculated by normalizing force to the pellet cross-sectional area, and equilibrium modulus was determined from the slope of the best-fit linear region of the equilibrium stress-strain relationship. The MicroTester measurements showed no statistically significant difference in equilibrium modulus among the TGF-β1-, FGF-2-, and dual-primed groups, although the combined TGF-β1/FGF-2 condition exhibited substantial donor-to-donor variability. These measurements provided a functional mechanical assessment of the engineered cartilage microtissues alongside biochemical, histological, and transcriptomic analyses.
