Primer sequences.

<p>Primer sequences.</p

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).</p

PTN induces hypertrophic differentiation of hBMSC.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in 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>): Alizarin red staining of chondrogenic pellets. (<b>B</b>): Collagen10 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>C</b>): Real-time polymerase chain reaction analysis of hypertrophic related genes expression. RNA were purified from hBMSC at day 0 and cultured without or with PTN at day 7 and 14. Expression levels of hypertrophic genes Matrix Metalloprotease 13 (MMP13), Collagen 10A1 (Col10) and Alkaline Phosphatase (ALP) 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 when they are different of 0. 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<0.01; ###: p<0.001) and between values from increasing PTN doses from the same day (*: p<0.05; **: p<0.01).</p

PTN increases cartilage specific protein and gene expression during hBMSC chondrogenic differentiation.

<p>hBMSC from 3 independent patients were cultured in micromass with chondrogenic medium in 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>): Collagen 2 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100 µm. (<b>B</b>): Real-time polymerase chain reaction analysis of cartilage related genes expression. RNA were purified from hBMSC at day 0 and after 7 and 14 days of culture without or with PTN. Expression levels of cartilage genes: SRY-box9 (Sox9), Cartilage Oligomeric Matrix Protein (COMP) and Collagen9A1 (Col9) are normalized 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<0.01; ###: p<0.001), and between values from increasing PTN doses from the same day (**: p<0.01; ***: p<0.001).</p

PTN chondroinductive effects are inhibited by inhibitors of PTN receptors and Pi3K.

<p>hBMSC were cultured in micromass with chondrogenic medium with 0/ml or 500 pg/ml of PTN with or without Ly294002 (15 µM) or p111-136 peptide (100 ng/ml). All conditions were performed in triplicate. (<b>A</b>): Real-time polymerase chain reaction analysis of late chondrogenic marker genes after 14 days of treatment. Expression levels of Matrix Metalloprotease 13 (MMP13), Collagen 10A1 (Col10) and Alkaline Phosphatase (ALP) are related to Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH). Values are the mean±SEM. Statistical analysis were performed, with a one way anova Kruskal-Wallis test between DMSO treated and Ly294002 or P111-136 treated hBMSC (*: p<0.05; ***: p<0.001). (<b>B</b>): Collagen10 immunostaining of chondrogenic pellets. Side box shows an enlargement (x3) of the black square. Bars represent 100µm.</p