The Effects of Tgf-beta1 and Igf-i on the Biomechanics and Cytoskeleton of Single Chondrocytes

Objective Ascertaining how mechanical forces and growth factors mediate normal and pathologic processes in single chondrocytes can aid in developing strategies for the repair and replacement of articular cartilage destroyed by injury or disease. This study examined effects of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor-I (IGF-I) on the biomechanics and cytoskeleton of single zonal chondrocytes. Method Superficial and middle/deep bovine articular chondrocytes were seeded on tissue culture treated plastic for 3 and 18 h and treated with TGF-β1 (5 ng/mL), IGF-I (100 ng/mL), or a combination of TGF-β1 (5 ng/mL) + IGF-I (100 ng/mL). Single chondrocytes from all treatments were individually studied using viscoelastic creep testing and stained with rhodamine phalloidin for the F-actin cytoskeleton. Lastly, real-time RT-PCR was performed for β-actin. Results Creep testing demonstrated that all growth factor treatments stiffened cells. Image analysis of rhodamine phalloidin stained chondrocytes showed that cells from all growth factor groups had significantly higher fluorescence than controls, mirroring creep testing results. Growth factors altered cell morphology, since chondrocytes exposed to growth factors remained more rounded, exhibited greater cell heights, and were less spread. Finally, real-time RT-PCR revealed no significant effect of growth factor exposure on β-actin mRNA abundance. However, β-actin expression varied zonally, suggesting that this gene would be unsuitable as a PCR housekeeping gene. Conclusions These results indicate that TGF-β1 and IGF-I increase F-actin levels in single chondrocytes leading to stiffening of cells; however, there does not appear to be direct transcriptional regulation of unpolymerized β-actin. This suggests that the observed response is most likely due to signaling cross-talk between growth factor receptors and integrin/focal adhesion complexes