Page 36 - Improved endothelialization by silicone surface modification and fluid hydrodynamics modulation- Implications for oxygenator biocompatibility Nasim

Iran) in 0.02 M acetic acid to immobilize collagen at 4°C for 24 h. Unbound collagen was removed by washing the collagen-immobilized silicone tubes with water. Collagen-adsorbed silicone (Si-Col), collagen-immobilized plasma surface- modified silicone (PSM Si-Col), collagen-immobilized AAc-grafted silicone (AAc Si- Col), and collagen-immobilized AmS-grafted silicone (AmS Si-Col) tubes were dried at room temperature and stored at 4°C before use. Some tubes were washed extensively twice with water to assess the stability of the collagen linked to the tubes. The amount of immobilized collagen on the tubes before and after washing was determined by a Bradford protein assay (Bradford, Hercules, CA, USA) using an Eppendorf biophotometer D30 (Eppendorf, Hamburg, Germany) following the manufacturer’s instructions [18]. Concentrations of immobilized collagen on modified silicone tubes were assessed by comparison with a standard curve.
Characterization of surface-modified silicone tubes
The effect of peroxide, carboxyl, and amine groups on mechanical properties of silicone tubes was evaluated by holding both ends of each Si, PSM Si, AAc Si, and AmS Si tube (length 9 mm) in a specific grip of an in-house fabricated uniaxial testing instrument, and pulling uniaxially until tension break. The ultimate tensile strength was determined based on the peak load and the initial surface area of each tube. The percent elongation-at-break was obtained from the ratio between the elongated length at the time of failure (l) and initial length (l0) of each silicone tube [32, 33].
The wettability of unmodified and surface-modified silicone tubes (i.e. Si, PSM Si, AAc Si, AmS Si, PSM Si-Col, AAc Si-Col, and AmS Si-Col) was probed by static water contact angle measurement [21, 34] using Kruss G10 goniometer contact-angle measurement equipment (Krüss GmbH, Hamburg, Germany). Tubes were cut longitudinally, glued on a microscope glass slide, and mean values of five water contact angle measurements on randomly chosen areas of each tube were calculated.
Endothelial cell seeding, adherence, and proliferation
Human umbilical vein endothelial cells (HUVECs) were obtained from the National Cell Bank, Pasteur Institute of Iran (Tehran, Iran), and used between passages 3 and 6 to evaluate cell attachment and proliferation on unmodified and surface- modified silicone tubes. Hundred μl endothelial cell suspension containing 300 cells/μl Dulbecco's modified Eagle's medium (DMEM)/F12 with 10% fetal bovine serum (Gibco, Renfrewshire, Scotland) was infused from one end into the lumen of each sterile tube using a syringe. During cell seeding for 4 h, the tubes were rotated every 30 min to promote homogeneous cell adhesion to the inner surface of the tubes. Cells were either cultured for 6 days in a humidified atmosphere of 5% CO2 in air at 37°C, with medium replacement every 2 days, or used to determine
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