Lentivirus-mediated knockdown of aggrecanase-1 and -2 promotes chondrocyte-engineered cartilage formation in vitro.

Chondrocyte-based tissue engineering has emerged as a promising approach for repair of injured cartilage tissues that have a poor self-healing capacity. However, this technique faces a major limitation: dedifferentiation of chondrocytes occurs following several passages in culture. Aggrecan, a major component of cartilage extracellular matrix, plays an essential role in chondrocyte differentiation. The aim of this study is to determine whether inhibition of chondrocyte aggrecanases, key degradative enzymes for aggrecan in cartilage, could benefit chondrocyte differentiation and the preservation of chondrocyte phenotype within a long-term period. Lentivirus-mediated RNA interference (RNAi) was employed to target both aggrecanase-1 and -2 in primary rat chondrocytes, and the transduced cells were seeded into chitosan-gelatin three-dimensional scaffolds. Histological, morphological, and biochemical analyses were performed at 1-8 weeks post-implantation to study chondrocyte survival, differentiation, and function. We found that lentivirus-mediated RNAi notably decreased the abundance of aggrecanase transcripts in chondrocytes but did not affect cell viability. Most importantly, compared to the control constructs seeded with untransduced chondrocytes, the aggrecanase inhibition increased chondrocyte proliferation and reinforced the production of glycosaminoglycans and total collagen, indicative of chondrocyte differentiation. The mRNA expression of chondrocyte marker genes (collagen II and aggrecan) was enhanced by aggrecanase silencing relative to the control. Together our data demonstrate that inhibition of endogenous aggrecanases facilitates chondrocyte differentiation and chondrocyte-engineered cartilage formation in vitro. The combination of lentiviral delivery system and genetic manipulation techniques provides a useful tool for modulation of chondrocyte phenotype in cartilage engineering.