Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes

Objective: When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGF beta) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGF beta supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. Methods: Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGF beta. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. Results: Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGF beta 3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. Conclusions: This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.