Page 35 - Improved endothelialization by silicone surface modification and fluid hydrodynamics modulation- Implications for oxygenator biocompatibility Nasim

Plasma pre-modification of silicone tubes
The surfaces of unmodified silicone tubes were cleaned three times with 70% (v/v)
ethanol, and once with water for 5 min. Unmodified silicone tubes were placed at
the bottom of a reaction chamber (Seren R600, Anatech ltd, Union City, CA, USA),
which was evacuated to 0.6 mbar, and pretreated with 60 W of oxygen plasma for
0.5 min. After plasma surface modification, silicone tubes were exposed to air for 5
min to generate peroxide groups on the surface ("plasma surface-modified silicone
(PSM Si) tubes"). Peroxide groups contacting air are more unstable and fade away
faster than peroxide groups contacting water [21]. Therefore PSM Si tubes were
either immediately (within 3 min) used for carboxyl and amine functionalization, or 3 stored up to 2 days in water for characterization tests.
Carboxyl functionalization of silicone tubes
PSM Si tubes were immersed in 30% AAc in water for 30 min at room temperature, air-dried at 40oC for 5 min, and placed in a reaction chamber for plasma graft copolymerization for 3 min to prepare AAc-grafted silicone tubes (AAc Si). The residual monomers and homopolymers were removed by 24 h incubation in water [20, 21, 28]. The grafted amount of AAc was calculated by a gravimetric method according to the following equation:
rafted amount ( g cm2
g0 A
Where Wg is the dry weight of grafted silicone tube, W0 is the dry weight of unmodified silicone tube, and A is the inner surface area of silicone tube [21].
Amine functionalization of silicone tubes
Aminosilanization of PSM Si tubes was carried out as described earlier [29, 30]. In short, PSM Si tubes were immersed in 5% APTES in chloroform for 12 h at room temperature under nitrogen gas. For hydrolytic stabilization of amine layers, the aminosilane-grafted silicone (AmS Si) tubes were submerged in water for 48 h before use in cell experiments. The grafted amount of AmS was also calculated by a gravimetric method [21].
Collagen immobilization on silicone tubes
AAc Si, and AmS Si tubes were immersed into 30 ml 5 mM 2-(N- morpholino)ethanesulfonic acid (MES) buffer solution containing 48 mg 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 15 mg N- hydroxysuccinimide (NHS; Fluka, Neu-Ulm, Germany) before collagen immobilization [31]. The solution was gently stirred for 5 h at 4°C to activate the functional groups on silicone tubes. Then Si, PSM Si, AAc Si with activated carboxyl groups, and AmS Si tubes with activated amine groups were filled with 1 mg ml−1 collagen (acid soluble collagen type I, Pasteur Institute of Iran, Tehran,
22
2