INTERNALISATION OF CALCIUM PHOSPHATE AND MAGNESIUM PHOSPHATE BIONS BY ENDOTHELIAL CELLS UTILISING SCANNING ELECTRON MICROSCOPY AND CONFOCAL MICROSCOPY

Aim of the study was to investigate whether endothelial cells internalise magnesium phosphate and calcium phosphate bions.

Material and methods. Magnesium phosphate and calcium phosphate bions synthesised through supersaturation of the culture medium with the respective salts were further added to the confluent cultures of primary human coronary artery endothelial cells for 4 h. Internalisation was evaluated by backscattered scanning electron microscopy (BSEM) and confocal microscopy. For BSEM, cells were fixed in 10% neutral phosphate buffered formalin, postfixed/stained by 2 % osmium tetroxide, dehydrated in a graded ethanol series, stained by 2% uranyl acetate, impregnated into acetone:epoxy resin (1:1) and into the fresh epoxy resin with its subsequent polymerisation, grinding, and polishing. Samples were then counterstained by lead citrate, sputter coated with carbon, and visualised utilising BSEM. Confocal microscopy was conducted after exposure of endothelial cells to FITC-labeled magnesium phosphate bions or calcein-labeled calcium phosphate bions with the following staining by a pH sensor LysoTracker Red and nuclear stain Hoechst 33342.

Results. All types of bions were internalised by endothelial cells within 4 h. BSEM after long-term postfixation/ staining by osmium tetroxide and uranyl acetate, embedding into epoxy resin and counterstaining by lead citrate visualised multiple black electron-dense mineral inclusions which were not detected both within the control cells and in the intercellular space. Confocal microscopy also detected internalised MPB and CPB in cytosol and lysosomes.

Conclusion. BSEM visualised MPB and CPB internalised by endothelial cells after 4 h exposure while confocal microscopy detected their colocalisation with lysosomes. MPB is an appropriate comparison group to study the specificity of CPB-related pathogenic effects as both of these bion types have close physico-chemical properties and are similarly internalised. 

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