Page 37 - Improved endothelialization by silicone surface modification and fluid hydrodynamics modulation- Implications for oxygenator biocompatibility Nasim

cell attachment at 4 h. The attached cells were washed with phosphate buffered saline (PBS), released with trypsin/ethylene diamine tetraacetic acid (EDTA; Merck, Kenilworth, NJ, USA), and stained with 0.4% trypan blue (Sigma-Aldrich, St. Louis, MO, USA) to determine viable cell number in a Neubauer cell chamber. The percentage of adherent cells was calculated from the cell counts upon seeding and after 4 h of culture [11, 22].
Endothelial cell proliferation on the surface-modified silicone tubes at days
2, 4, and 6 was estimated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) as described elsewhere [9, 12, 33]. The absorbance was measured at 545 nm using 3 an ELISA Reader (Stat Fax-2100, Miami, FL, USA). The number of endothelial
cells was quantified using a calibration curve with known cell numbers.
Endothelial cell morphology
The morphology of endothelial cells attached to either unmodified or surface- modified silicone tubes was visualized after 6 days culture by scanning electron microscopy (SEM) using an Essen Philips XL 30 ESEM Environmental electron microscope (Philips, Amsterdam, The Netherlands). The tubes were cut longitudinally to allow observation of cell morphology in the lumen of cell-seeded silicone tubes. Tubes with adherent cells were rinsed with PBS, fixed with 4% (v/v) glutaraldehyde solution in PBS at 4°C for 30 min, washed once with ultrapure water, dehydrated in a series of ethanol/distilled water solution (10% ethanol increments; each step 3 min), and finally dried at room temperature [35]. The optimum parameters for SEM imaging were 20 kV electron accelerating voltage, 15 mm working distance, and 500x magnification.
Fluid shear stress treatment of endothelialized silicone tubes
To compare the different surface-modified silicone tubes for their ability to support cell attachment under fluid shear stress, an in-house designed and fabricated circulating flow loop was used. After 6 days of culture, tubes with adhered cells were washed with PBS and mounted on a holder. Endothelial cells on silicone tubes were exposed to a fluid shear stress of 1.5 N m-2 for 1 h using a peristaltic pump (Heidolph, Schwabach, Germany). Polyvinyl chloride tubing was used to link the medium reservoir, pump, and silicone tube. Three-way valves were used to stop entering fluid into the silicone tubes at the end of an experiment (Figure 1). The percentage of detached cells was assessed by detracting the number of cells after shear conditioning from the number before shear conditioning using the MTT assay.
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