Page 127 - Tailoring Electrospinning Techniques for Regenerative Medicine - Marc Simonet

hematoxylin and eosin (HE) and Sirius red F3B (Gurr BDH, 34149) in saturated picric acid solution. Immunohistochemical stainings were performed a er depara inization and dehydration. Sections were stained for alpha-smooth muscle actin for myofibroblasts and smooth muscle cells (1:32.000 anti-a-SMA, Sigma A2547). Antibodies were diluted in PBS/BSA/Azide. A er depara inization, the primary antibody was added for 1 hour at room temperature. Slides were subsequently incubated with poly-AP goat anti-mouse IgG (Brightvision, DPVM110AP) for 30 min at room temperature. Binding of the secondary antibody was visualized with liquid permanent red reagent. For quantative analyses, two investigators who were blinded for time point of explantation, conducted quantative analysis. Sections were photographed (NikonE800 microscope with ACT-1 so ware) and on the HE stained slides, cellularity was studied under a 20x magnification and a high power magnification (high power field (hpf), 40x objective lens). Per valve leaflet the total number of 4 areas were manually counted using ImageJ so ware. The amount of collagen was analyzed with a picosirius red staining and polarized light. The area of collagen on photographed sections were digitally converted into binary images. The average grey area of 4 hpfs was calculated and normalized to the total tissue area in each section. The mean of the measured fields was calculated for each sample.
The mechanical properties of the explanted tissues were performed on sample strips of 15*3 mm at 100%/min and a sample gauge length of 10 mm. Due to early degradation not every valve leaflet could be used for mechanical analysis, resulting in n=1 valve a er 2 weeks, n=2 a er 3 weeks, n=4 a er 4 weeks and n=1 a er 5 weeks. Per valve 1 to 3 samples were analyzed and averaged per valve.
To record the molecular weight, samples of the valves were treated with Clorox to remove the cells and the remaining PCL2kU4Un material was analyzed by GPC.
6.3.4 Statistical analysis
Histological and mechanical data are presented
as averages and their standard deviations (SD).
Statistical analyses on the mechanical data prior to
the implantation were performed using an unpaired
t-test using Graphpad Prism. Di erences were
considered significant for p values <0.05. 6 Statistical analyses for changes over time in
cellularity, collagen content, and mechanical properties were performed with linear regression (Gaussian distribution assumed). Changes in cardiac output and mean pressure during functionality testing were analyzed with a non-linear regression (one-phase decay model) and linear regression when non-linear regression could not fit (Gaussian distribution assumed). A decrease of 0-10% was considered not relevant.
FROM A POLYMER TOWARDS AN IN-SITU TISSUE ENGINEERED HEART VALVE
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