Mitochondrial assays materials

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

Immunological methods for enzymes, more specifically isoenzymes, such as lactate dehydrogenase-1 (167, 168), mitochondrial aspartate aminotransferase (169), prostatic acid phosphatase (170, 171,172), and creatine kinase-MB (173, 174, 175), have been in use in the clinical laboratory for 10 years. However, the use of the immunological rather than catalytic properties of enzymes has not provided the opportunities for standardization that was anticipated a number of years ago (176, 177, 178). It is only within the last year that a working group on CK-MB mass assay was formed under the auspices of the Standards Committee of the American Association for Clinical Chemistry (AACC). The objective of this working group is to prepare a reference material to calibrate methods that are based on the principle of CK-MB mass measurement. 

Cell culture testing is used widely in vitro to evaluate the biocompatibihty of HA samples. For this, several osteoblast cell lines have been developed to assess biomaterials performance in vitro, including a murine osteoblastic cell hne (MC3T3-El) [121], a human osteoblastic precursor cell line (OPCl) [122] and a human osteoblast cell line (hFOB 1.19) [123]. Osteoblast cells interact differently with material structures, depending on the combinations of chemical, structural and environmental variables. The biocompatibility of HA surfaces can be assessed using ceU-material interaction studies such as attachment, proliferation and differentiation. Cell proliferation on HA samples is normally investigated with a mitochondrial (MTT) assay, which permits the quantitative estimation of the number of living cells on a material. 

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