Various in vivo and in vitro studies suggest that joint homeostasis may have a crucial effect on the quality of regeneration tissue resulting from cartilage tissue engineering techniques. The goal of the current study was to evaluate the effect of synovial fluid (SF) from injured knee joints on in vitro chondrogenesis. Chondrocytes were isolated from a healthy human femoral condyle (post-mortem) and expanded in monolayer for 2 passages. Subsequently, the chondrocytes were redifferentiated for 14 days on collagencoated filters, cultured either in the presence or absence of 10% SF. SF was obtained from 12 injured human knee joints. After 14 days of culture, SF supplementation resulted in a significant downregulation of final proteoglycan (PG) content (7.3 +/- 1.8 mg versus 15.6 +/- 1.3 mg; p = 0.0001), PG content normalized to DNA (0.7 +/- 0.5 mg/microg versus 3.0 +/- 0.6 mg/microg; p < 0.05), relative collagen type II mRNA levels normalized to GAPDH mRNA levels (0.2 +/- 0.3 versus 7.0 +/- 5.6; p < 0.001), and differentiation index (collagen type II/I mRNA ratio; 0.1 +/- 0.2 versus 6.0 +/- 2.9; p < 0.001) as compared to control culture conditions. Additionally, SF-supplemented media resulted in significantly increased cellularity, as reflected by DNA content, compared with control media (1,369 +/- 683 microg versus 514 +/- 72 microg; p < 0.0001). Morphology, and collagen type I, X, and aggrecan mRNA levels were not significantly affected. In conclusion, this study demonstrates that SF from injured human knee joints significantly affects in vitro chondrogenesis and therefore may provide a viable target for future improvement of ACT by refinement of culture techniques, patient selection, or pretreatment of affected joints to restore joint homeostasis.