Computational protocol: Simvastatin and atorvastatin reduce the mechanical properties of tendon constructs in vitro and introduce catabolic changes in the gene expression pattern

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Protocol publication

[…] Tensile testing of the constructs was performed after 7 days of statin treatment (21 days after cell seeding). The testing was performed in a PC-driven micromechanical rig with a liquid chamber (20 N load-cell, sampling rate 10 Hz; Deben, Suffolk, UK). A stereoscopic microscope (SMZ1000, Nikon, Tokyo, Japan) with C-mount lens (8x), equipped with a 15 Hz digital camera (DFWX700, Sony, Tokyo, Japan; 640x480 Pixel) was used for imaging during the test to verify clamping length and monitor the rupture site of the construct. The tendon constructs were glued on specimen plates with a mounting distance of 12.5 mm. The specimen was thereafter transferred to a cell culture medium-bath and after a short adaptation period, the test was started. The samples were stretched at 4 mm/min until failure (). Construct diameter and mounting length were measured before the stretching started by capturing images though the microscope and calculating the distances using ImageJ (NIH, USA). The diameter was measured four different places (the thickest and thinnest places on the right and left side of the construct using 4x objective magnification) and an average cross sectional area was calculated assuming a circular cross-section. The length of the samples was also measured at 0.8x objective magnification. Force was filtered by a running average over 10 data points (equal to 1 s or ~0.5% strain) before calculating stress based on the cross sectional area. Strain was determined from the length at the onset of force (point where stress first exceeds 10 kPa). Tensile modulus was calculated as the peak slope determined by linear regression over a 1% strain range. The mechanical testing was performed in triplicates for each cell line and a mean value for each parameter across these triplicates was used for further analysis. For technical reasons, three of the samples only had duplicate mechanical measurements. The mean for each cell line treated with statins was thereafter used for statistical analysis. [...] The mechanical data, the hydroxyproline assay and the MTT assay were first tested with a Wilcoxon matched-pairs signed rank test to test if there were any statistical difference between DMSO-controls and untreated-controls. The treatment effect of statins was thereafter analyzed with a repeated measures Friedman test followed by Dunn´s test for multiple testing against the DMSO-controls. These statistical analyses were done in GraphPad Prism.Log transformed gene expression data was first analyzed by comparing if there was any difference between DMSO-controls or untreated-controls with a repeated measures 2-way ANOVA with the day 1 and 7 data from DMSO-controls and untreated-controls. When no difference was found we continued to normalize all the statin treated samples to the same day DMSO-control from each cell line and thereafter analyzed the data again with a repeated measures 2-way ANOVA with time and treatment as independent variables. This was followed by Dunnett´s method for multiple comparisons against the DMSO-controls. One sample in the untreated-control group (day 1) was excluded from analyses on all targets due to an extreme deviating result. The MMP-1 expression was more than 400 standard deviations from the mean after normalization within the cell line. Also the expression of TIMP-1, MMP-9 and scleraxis had all greater values than 2 standard deviations from the mean and there was no detectable expression of tenomodulin. This exclusion was done before the statistical analyses were performed. p<0.05 was considered significant. These statistical analyses were done in SigmaPlot. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Rupture, Tendinopathy
Chemicals Simvastatin