Scanning electron microscopy methodology for study of the pathophysiology of calcification in bioprosthetic heart valves.

Scanning electron microscope (SEM) morphologic analysis combined with energy dispersive characteristic X-ray (EDX) microprobe analysis provides insight into the mechanisms associated with disease-related crystal formation in biological materials. SEM and EDX were employed in analyzing specimens which were embedded in standard fashion in glycolmethacrylate. The specimen surfaces under electron microscope investigation resulted from microtomy used in the preparation of reference light microscope histological sections; thus histology served as a direct reference for the SEM and EDX analyses. The particular application of these methods was in the study of bioprosthetic heart valve calcification, largely responsible for clinical failure of these heart valve substitutes. To simulate the clinically observed mineralization processes, glutaraldehyde-pretreated porcine heart valve leaflets were implanted subcutaneously in rats and subsequently removed at various time intervals from 1 to 56 days. Also, to address the hypothesis that the calcification process generates crystalline materials analogous to those in bone, EDX data obtained from pure hydroxyapatite were compared with the embedded tissue results. Further, EDX results were compared with data obtained by chemical analysis of the bulk specimens to assess the validity of the electron microscope technique.