Development of a sensitive whole blood chemiluminescence method for assessing the bioactivity of calcium phosphate powders

Abstract

The development of simple, but highly sensitive and accurate in vitro methods for assessing the bioactivity of biomaterials is a key requirement for assuring their successful clinical application. Moreover, screening techniques that will allow various materials to be recommended, or otherwise, for further testing, have the potential to make appreciable savings in the time and expense associated with product development. This paper gives details of a novel whole blood chemiluminescence method, based on the photoprotein Pholasin ®, which emits light in the presence of the free radical superoxide, other reactive oxygen species and the enzyme myeloperoxidase. This whole blood assay may have the potential as a screening method for assessing calcium phosphate (Ca-P) bioceramic powders according to their relative bioactivity. The method employed involves exposure of samples of anti-coagulated (heparinised) blood to the various Ca-P materials and stimulating the contacted leukocytes in the blood with phorbol-12-myristate-13-acetate (PMA). PMA stimulates protein kinase C directly and sets in motion a cascade of events in the leukocytes leading to the activation of the superoxide generating NADPH oxidase system. PMA also promotes degranulation, which is the fusion of the granule membrane with the plasma membrane and the extracellular release of granule enzymes. Changes in the free radical and degranulation activity of the cells, as measured by the luminescent response of Pholasin ® to cells stimulated with PMA, after exposure to the Ca-P powders, are presented as percentage differences from the response to (control) blood samples not exposed to the powders. The resultant data clearly indicate significant differences in the light detected via Pholasin ® from the blood exposed to the bioceramic materials compared to that from the relevant controls. These differences correlate well with the ascribed bioactivity of the Ca-P powders, as assigned from the measured chemical and structural properties. Furthermore, the method can also be applied to samples of blood, which have been diluted in the ratio 1:100 in standard blood dilution buffer. The light output response in the diluted blood experiments is exactly the same as that observed with undiluted whole blood but occurs immediately as compared to the many minutes required to give response in the latter case. Thermally processed (800°C) Ca-P materials have also been studied. A relationship between the heat-induced properties of the powders and the chemiluminescence responses recorded from blood cells stimulated with PMA has been established.