Interactions between whole blood and biomaterial surfaces. With focus on the respiratory burst of adhering neutrophils as an early sign of cytotoxicity

Abstract

Although a vast array of materials is used to augment or replace a natural function in the human body, many of them cause side-effects or may be rejected. The growing need for more biocompatible devices has been accompanied by an increase in new materials. For evaluation of such new materials, it is of importance to develop simple, rapid and sensitive in vitro models. Therefore, one aim of this thesis was to develop a model for estimating the toxicity of fluid materials, based on the degree of diminution of the respiratory burst activity of neutrophils. During the respiratory burst, oxygen is reduced by NADPH oxidase to reactive oxygen species. The effects of commercially used heat-sterilized peritoneal dialysis (PD) fluids on the respiratory burst response of titanium-adhering neutrophils were compared with the effects of filter-sterilized PD fluids. Filter-sterilized PD fluids lack the toxic components that develop during the commercial heat sterilization of PD fluids. The possibility of reducing the toxic effect of heat-sterilized PD fluids by addition of scavengers/antioxidants was also investigated.Bleeding and inflammation are the first steps in wound healing around biomaterials. Understanding each step of wound healing is of importance for understanding the whole process. Another aim of this thesis was therefore to study the interactions between whole blood and biomaterial surfaces. Blood cell adhesion to hydrophilic and hydrophobic glass surfaces was studied. The kinetics of formation of the three key proteins thrombin, kallikrein and complement C5b-9 on hydrophilic and hydrophobic glass and titanium surfaces was also investigated. Neutrophils adhering to titanium surfaces after whole blood exposure for 1 hour were found to be in a primarily activated state, since the expression of surface integrins was down-regulated and the expression of selectins was not detected. They also mounted a respiratory burst in the presence of stimulating agents. The respiratory burst response was attenuated in a time-dependent manner for neutrophils incubated in heat-sterilized PD fluids, while the response for neutrophils in a control buffer and in filter-sterilized PD fluids was not affected. The addition of glutathione to PD fluids improved the extracellular respiratory burst activity, whereas the intracellular activity was not affected.Thrombin and kallikrein were generated on hydrophilic and hydrophobic glass and titanium surfaces after a few seconds, and C5b-9 was detected after 32 minutes of blood exposure. Platelets were found on glass surfaces after a few seconds and were followed by neutrophils after 8 minutes, lymphocytes after 32 minutes and monocytes after 2 hours. Platelet adhesion was faster to hydrophobic glass. Activation of the coagulation cascade on titanium and glass was higher on the hydrophilic surfaces. C5b-9 coverage showed a different pattern on each surface and became denser with time.In conclusion, initial interactions at blood-material interfaces are dependent on both the type of material used and its surface properties. Furthermore, titanium-adhering neutrophils constitute a simple and sensitive model for cytotoxicity testing of fluid materials such as PD fluids.