Enhanced cartilage formation by inhibiting cathepsin K expression in chondrocytes expanded in vitro

Although engineered cartilage has great potential in cartilage regeneration and reconstruction, dedifferentiation of chondrocytes during in vitro expansion remains a technical bottleneck in the clinical application. To overcome the problem, a gene modification approach was developed to knock-down the key gene involving dedifferentiation of human chondrocytes. A microarray assay revealed 84 up-regulated genes and 56 down-regulated genes in passage 4 (dedifferentiated) human chondrocytes compared to passage 1 cells. Among them, cathepsin K (CTSK) was the key gene (with 28 folds of increased gene expression), which was further confirmed by RT-PCR and Western-Blot. Furthermore, over-expression of CTSK led to reduced matrix production in cultured human chondrocytes in vitro and poor formation of engineered cartilage in vivo. In contrast, CTSK knock-down could better maintain the chondrogenic phenotype of in vitro expanded cells with increased gene and protein expression of collagen II and aggrecan when compared to control cells. More importantly, after 6 passages, the knock-down cells formed much better engineered cartilage than the control cells after in vivo implantation with 30% Pluronic F127 for 8 weeks as the experimental group formed much bigger sized cartilages with significantly increased weight and glycosaminoglycan content (p < 0.05) than the control group. Histologically, the knock-down cells formed a more homogenous cartilage structure with enhanced production of collagen II and proteoglycans. Overall, these results suggest that CTSK knock-down may provide a feasible way to expand functional human chondrocytes in vitro for engineering good quality human cartilage and thus may have its great potential in the clinical translation of engineered cartilage in the future, given the fact that biosafe RNA interference techniques are already available.