Scanning electron microscopy and energy-dispersive x-ray microanalysis: a valuable tool for studying cell surface antigen expression on tissue-engineered scaffolds.

The influence of an acellular porcine matrix on proinflammatory activation of endothelial cells (EC) during normoxia and hypoxia was investigated by a newly established semi-quantitative electron microscopic procedure. As a model, three adhesion molecules (E-selectin, ICAM-1, and VCAM-1) were localized by silver-enhanced immunogold staining and energy dispersive X-ray microanalysis after normoxic or hypoxic pretreatment of the cells and subsequent stimulation with IL-1beta. Morphology of EC grown on porcine matrix or coverslips was recorded simultaneously using secondary electron imaging. EC appeared tightly attached to the underlying surfaces with their typical cobblestone-like morphology. Statistically significant upregulations upon stimulation with IL-1beta were observed in both groups for all three adhesion molecules. Hypoxic pretreatment of the specimens with subsequent reoxygenation neither induced morphological changes nor caused an upregulation of adhesion molecule expression in cells grown on acellular porcine tissue. Unexpectedly, in cells seeded onto the acellular matrix, IL-1beta failed to upregulate ICAM-1 expression after a short period of hypoxia. The surface expression of VCAM-1 was also significantly lower even under normoxic conditions, which might indicate the development of functional impairment of cells in contact with acellular porcine tissue. The method presented in this study has proven valuable for the determination of antigen expression on scaffold materials in parallel with the characterization of surface morphology.