Figure 6
Effect of culture conditions on the protein expression of type I and II collagens.
(A) Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were cultured in collagen sponges at 21% O2 for 14 days, in incomplete chondrogenic medium in absence (control) or presence of 50 ng/ml of BMP-2 and 10 ng/ml of TGF-β1. Undifferentiated UCB-MSCs were also cultured as monolayers and used as a control before differentiation (day 0, D0). (A) Protein extracts were analyzed in Western blots for type II and type I collagens versus GAPDH. Representative blots are shown (n = 6). Human articular cartilage (HAC) shows different levels of type II collagen maturation forms such as type II procollagen (pro), with only C- or N- terminal propeptides (pC/pN) and the doubly cleaved form (mature form). (B) Sponge constructs were obtained as described in panel A. hUCB-MSCs in sponges were fixed, permeabilized, and immunostained with anti-type II and anti-type I collagen rabbit antibodies. Goat anti-rabbit Alexa Fluor 546 secondary antibodies were used to visualize the localization of type I and II collagens (red). Nuclei were counterstained with DAPI (blue). Pictures were taken on a confocal laser-scanning microscope. Scale bar: 50 μm. For each collagen isotype left panels show fluorescence staining and the right panels, fluorescence staining merged with the corresponding light transmission image.
