PTN increases glycosaminoglycans content of chondrogenic induced hBMSC.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in the absence or with increasing doses of PTN (0 pg/ml white boxes, 50 pg/ml grey boxes and 500 pg/ml black boxes) for 21 days. All conditions were performed in triplicate per patient. (<b>A</b>): Alcian blue staining of sulfated GAGS in chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>B</b>): Total sulfated GAGs quantification. After 14 days in micromass culture, sulfated GAGs were extracted from pellets and quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088287#pone.0088287-Barbosa1" target="_blank">[27]</a>. GAG amount was normalized according to pellet volume and reported as µg of total GAGs per mm³. (<b>C</b>): Real-time polymerase chain reaction analysis of proteoglycan protein core-gene expression. RNA were purified from hBMSC at day 0 and after 7 and 14 days of culture without or with PTN. Expression levels of proteoglycans core expression Aggrecan (ACAN), Biglycan (BGN), Decorin (DCN), Versican (VCAN) are related to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. The gene expression values at day 0 are represented by straightened lines. Statistical analysis were performed, with a one way anova Kruskal-Wallis test, between values from free-PTN conditions (white boxes) at day 0, 7 and 14 (###: p<0.001), and between values from increasing PTN doses from the same day (*: p<0.05; **: p<0.01; ***: p<0.001).