In vitro study of chondrocyte redifferentiation with lentiviral vector-mediated transgenic TGF-β3 and shRNA suppressing type I collagen in three-dimensional culture.

Chondrocytes are the primary candidate therapeutic cells to cure cartilaginous lesions. Ideally, for transplantation, autologous chondrocytes are isolated from the patient, amplified in vitro, seeded in a scaffold and implanted back. However, significant concerns arise with chondrocyte dedifferentiation during monolayer amplification, whereby cells lose their chondrocytic phenotype by rapidly downregulating the expression of cartilage markers such as type II collagen (Col II) and aggrecan. The accompanying upregulation in type I collagen (Col I) is also problematic, as it leads to unexpected fibrosis and causes such engineered cartilage to lack the desired mechanical strength to make up joint lesions. Transforming growth factor-β3 (TGF-β3) has been proved effective in maintaining chondrocytic morphology and promoting total collagen production. In this study, we aimed to deliver the TGF-β3 gene into dedifferentiated chondrocytes with recombinant lentiviral vectors; by transgenic expression of TGF-β3, chondrocytic redifferentiation is catalysed. Simultaneously, shRNA targeting Col I was also incorporated into the vector to suppress Col I production. The results indicated that chondrocytes underwent dedifferentiation in monolayer culture in the presence or absence of transgenic TGF-β3. In three-dimensional culture, effective redifferentiation was managed in the dedifferentiated chondrocytes that were transduced with transgenic TGF-β3. The incorporation and expression of Col I-targeting shRNA were also effective in reducing Col I production in a post-transcriptional manner.