FIG. 4.

Chondrogenic differentiation of hBMSCs in an osteochondral environment in vitro is not solely mediated by TGFβ. (A) Collagen type 2 and aggrecan gene expression relative to GAPDH of hBMSCs from two different donors cultured for 28 days in alginate beads with or without TGFβ and anti-TGFβ or in simulated subchondral defects with or without anti-TGFβ (n=6 per MSC donor, mean with standard deviation, Kruskal–Wallis test followed by the Mann–Whitney U test); (B) TGFβ secretion of osteochondral biopsies cultured with or without anti-TGFb measured in medium used for 72 h (n=3, mean with standard deviation); (C) Collagen type 2 and aggrecan gene expression relative to GAPDH of hBMSCs from two different donors cultured for 28 days in alginate beads with or without TGFβ or in simulated subchondral defects without TGFβ. Samples were cultured with the pSmad2/3 inhibitor SB-505124 or the pSmad1/5/8 inhibitor DM (n=6 per MSC donor, mean with standard deviation, Kruskal–Wallis test followed by the Mann–Whitney U test); (D) TGFβ secretion of osteochondral biopsies measured in medium used for 72 h with or without the pSmad2/3 inhibitor SB-505124 or the pSmad1/5/8 inhibitor DM (n=3, mean with standard deviation); (E) GAG production relative to DNA concentration of hBMSCs from one donor cultured in alginate beads with or without TGFβ or in alginate in simulated subchondral defects without TGFβ. Samples were cultured with anti- TGFβ, SB-505124, or DM (n=6, mean with standard deviation, Student's t-test); (F) western blot for α-tubulin and phosphorylated Smad 2 (pSmad2) on hBMSCs cultured for 4 days in alginate beads or in alginate in simulated subchondral defects harvested after 1.5 h of stimulations with or without TGFβ, anti-TGFβ, and/or SB-505124. DM, dorsomorphin. Color images available online at www.liebertpub.com/tea