Endothelial cell seeding onto various biomaterials causes superoxide-induced cell death.

The seeding and/or in-growth of endothelial cells on a number of blood-contacting implants are a concern for both biomaterials and tissue engineering. While endothelialization has been viewed positively, owing to their ability to regulate both smooth muscle and blood, there is evidence which suggests that endothelial cells on a nonoptimized surface may be counterproductive. The present study describes the experimentation designed to elucidate the effect of culture substrate on intracellular superoxide (SO) levels, a marker for endothelial cell dysfunction. The adaptation of the use of dihydroethidium under physiologically relevant shearing conditions is also reported. The present study describes a standardized method for the use of dihydroethidium as a marker for intracellular oxidative stress under physiologic shear. Levels of hydrogen peroxide (oxidative stress producing agent) are optimized to a minimum of 60 microM (under static conditions) to allow for the detection of SO within the free radical scavenging environment. A flow rate of 24.4 mL/min is applied and found to produce physiologically relevant shear stress (8.2 dynes/cm(2)) within the system under study. Dihydroethidium is a useful marker for assessing intracellular oxidative stress in studies that require shear.