Low Agarose Concentration and TGF-β3 Distribute Extracellular Matrix in Tissue-Engineered Cartilage

Background: The mechanical properties of articular cartilage are dominated by the interterritorial matrix, as the matrix in this region is stiffer, greater in volume, and more interconnected compared to that in the pericellular and territorial region. Hence, tissue-engineered constructs in which a newly synthesized matrix accumulates in the pericellular and territorial regions may be of a lower mechanical quality compared to constructs in which the interterritorial region contains abundant matrix. Objective: In this study, we explored the extent to which matrix distribution may be modulated by altering the agarose concentration and the presence of the transforming growth factor-beta (TGF-beta) and how this affects the mechanical properties of cultured cartilage constructs. Methods: Cartilage development in constructs with agarose concentrations varying from 1%, 2%, and 3% (study 1) and in constructs with no or very low agarose concentrations of 0.25%, 0.5%, and 1% (study 2) were compared. In both studies, the effect of TGF-beta 3 was compared to fetal bovine serum. After 21 and 42 days of culture, the matrix content and distribution were analyzed and mechanical properties were assessed at day 42. Results: Culture in lower agarose concentrations did not significantly influence the matrix content per wet weight, but did result in a more homogeneous distribution. Constructs cultured with less agarose also showed a higher equilibrium modulus. The presence of TGF-beta 3 resulted in an increased extracellular matrix (ECM) deposition, a more homogeneous matrix distribution, and an equilibrium modulus. Conclusions: Culturing with no or low agarose concentrations and TGF-beta 3 is favorable for cartilage tissue-engineering studies, because both stimulate the formation of a more homogeneous ECM and consequently result in improved mechanical properties.