Cartilage tissue is avascular with less regeneration potential and therefore, cartilage regeneration is still a major challenge for therapeutic approaches. Commonly used treatment strategies involve the transplantation of autologous chondrocytes into the defect. Before that, it is required to increase the cell number in vitro resulting in unwanted chondrocyte dedifferentiation. This could impair subsequent tissue regeneration. Both growth factors TGF-ß1 and IGF-1 are used as strong inducer of chondrogenic redifferentiation, however, a controlled application of TGF-ß1 is essential to avoid adverse effects. Therefore, in the present study, we investigated the time-dependent influence of TGF-ß1 administration on chondrocyte redifferentiation.
Human chondrocytes were embedded in alginate and cultured in serum-free DMEM containing ascorbic acid, dexamethasone, ITSTM and IGF-1. TGF-β1 was supplemented for 3, 7 and 21 days. Afterwards, cell viability and synthesis of extracellular matrix (ECM) proteins was analyzed by histological staining.
Live/dead staining of chondrocytes incubated with TGF-β1 for 21 days displayed an enhanced proliferation and formation of cell clusters resulting in excessive outgrowth of fibroblastic-like cells. However, exposure to TGF-β1 over only 7 days caused also cell clustering with moderate cell proliferation. Additionally, after 21 days of cultivation proteoglycan synthesis was identified by alcian blue staining after both TGF-β1 supplementation for 21 and also 7 days. Aggrecan was also detected in the periphery of the cell clusters after TGF-β1 incubation for only 7 days. Chondrocytes lacked proteoglycan expression after three-day TGF-β1 administration.
We could show, that prolonged administration of TGF-β1 results in massive proliferation of chondrocytes which is accompanied by cell outgrowth. We found that TGF-ß1 exposure for seven days is sufficient for achievement of cell clustering without excessive cell proliferation, which is important for inducing subsequent chondrogenic differentiation. Results indicate that even an initial TGF-β1 administration could be sufficient for inducing chondrocyte proliferation and differentiation in vitro.