Scaffoldless Tissue-Engineered Cartilage for Studying TGF-β-mediated Cartilage Formation

Reduced TGF-β signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach to treating OA is to identify factors that act downstream of TGF-β’s anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (1) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (2) validate the system for identifying downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model since Sox9 is known to promote cartilage formation and since TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissue. An Alk5/TGF-β Type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation, including reduced thicknesses and reduced proteoglycan staining compared to control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.