Gene expression analysis: Effect of treatment on GAPDH, heat shock proteins and tendon related genes.

<p>Gene expression after 1 or 7 days of statin treatment. The samples were analyzed for GAPDH, heat shock proteins and tendon related genes. Low dose (L) statin treatment corresponds to 0.05μM of either simvastatin or atorvastatin and high dose (H) corresponds to 0.5μM. The DMSO-control is set as baseline, and mRNA expression in constructs treated with statins are shown relative to DMSO-control for each cell line and day. Data is presented on a logarithmic y scale as geometric means ± 95% confidence interval (CI). Individual donor-specific cell lines are represented by dots. Significant interaction in the repeated measures 2-way ANOVA (time*treatment) are below each graph.</p

Gene expression analysis: Effect of treatment on collagen, MMPs and TIMP-1.

<p>Gene expression after 1 or 7 days of statin treatment. The samples were analyzed for collagens, MMPs and TIMP-1. Low dose (L) statin treatment corresponds to 0.05μM of either simvastatin or atorvastatin and high dose (H) corresponds to 0.5μM. The DMSO-control is set as baseline, and mRNA expression in constructs treated with statins are shown relative to DMSO-control for each cell line and day. Significant changes from DMSO-control are indicated by * (p<0.05), ** (p<0.01), and *** (p<0.001). Data is presented on a logarithmic y scale as geometric means ± 95% confidence interval (CI). Individual donor-specific cell lines are represented by dots. Significant interaction in the repeated measures 2-way ANOVA (time*treatment) are indicated below each graph.</p

Gene expression analysis: Effect of treatment on GAPDH, heat shock proteins and tendon related genes.

<p>Gene expression after 1 or 7 days of statin treatment. The samples were analyzed for GAPDH, heat shock proteins and tendon related genes. Low dose (L) statin treatment corresponds to 0.05μM of either simvastatin or atorvastatin and high dose (H) corresponds to 0.5μM. The DMSO-control is set as baseline, and mRNA expression in constructs treated with statins are shown relative to DMSO-control for each cell line and day. Data is presented on a logarithmic y scale as geometric means ± 95% confidence interval (CI). Individual donor-specific cell lines are represented by dots. Significant interaction in the repeated measures 2-way ANOVA (time*treatment) are below each graph.</p

Collagen quantification.

<p>Total collagen content in tendon constructs with or without statin treatment for 7 days. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. The line represents median value. Individual donor-specific cell lines are represented by the six different symbols. The collagen content was approximately 10% of the construct dry weight. High dose treatment with atorvastatin reduced the collagen content compared to DMSO-controls, this was not seen after simvastatin treatment. Significant changes from DMSO-control are indicated by ** (p = 0.01).</p

PCR primers.

<p>PCR primers.</p

Construct mechanical data.

<p>Maximum force, maximum stiffness, cross sectional area, maximum stress and maximum modulus of tendon constructs with or without statin treatment for 7 days. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. Significant changes from DMSO-control are indicated by * (p<0.05), ** (p<0.01) or *** (p<0.001). The line represents median value. n = 6 different donor-specific cell lines, where each symbol represent the mean value from three replicates from one cell line. The force, stiffness, stress and modulus were all reduced after 7 days of high dose simvastatin treatment. High dose treatment with atorvastatin also reduced the force and stiffness and tended to reduce the maximum modulus (p = 0.07).</p

Mechanical data.

<p>Mechanical data.</p

Cell proliferation.

<p>Cell proliferation in tendon constructs, with or without statin treatment for 7 days, measured by a MTT assay. The data is presented as optical density (OD) value. Low dose (L) statin treatment corresponds to 0.05μM (simvastatin or atorvastatin) and high dose (H) corresponds to 0.5μM. The line represents median value. Individual donor-specific cell lines are represented by the six different symbols. The cell proliferation was reduced in constructs treated with high dose simvastatin or high dose atorvastatin. Significant changes from DMSO-control are indicated by * (p<0.05).</p

Summary of mRNA expression of tendon-related genes as a response to changes in the spatial/mechanical environment and TGF-β1 supplementation.

<p>Summary of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086078#pone-0086078-g002" target="_blank">figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086078#pone-0086078-g003" target="_blank">3:</a> The effect of the intervention is compared to the gene expression of the respective target cultured for six days under static tension, the arrows (↑↓) indicate significant changes relative to the tensioned samples, or no statistical change (↔). Arrows in parenthesis indicate a tendency (p< 0.1). Tenomodulin (TNMD); Scleraxis (SCX); Mohawk homeobox (MKX); Collagen type I (COL1A1); collagen type III (COL3A1); collagen type XII (COL12A1); collagen type XIV (COL14A1); Fibronectin (FN); Decorin (DCN); Fibromodulin (FBMD); Transforming growth factor-β1 (TGFB1); Connective tissue growth factor (CCN2); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).</p

Comparison between 2D monolayer and 3D tendon-constructs under static tension.

<p>The 3D tendon-construct is set as baseline, and mRNA expression of tendon cells grown on 2D monolayer is shown relative to the 3D tendon construct. Significant changes are indicated by *. Data presented on a logarithmic y scale baseline as geometric means ± SEM with 3D tendon-construct as baseline (n = 5).</p