Role of glutaraldehyde in calcification of porcine aortic valve fibroblasts

Abstract

Glutaraldehyde-treated porcine aortic valve xenografts frequently fail due to calcification. Calcification in the prostheses begins intracellularly. In a previous study, various types of cell injury to canine valvular fibroblasts, including glutaraldehyde treatment, led to calcification. An influx of extracellular Ca2+ into the phosphate-rich cytosol was theorized to be the mechanism of calcification. To test the Ca2+ influx theory, cytosolic Ca2+ and Pi concentrations were assessed in glutaraldehyde-treated porcine aortic valve fibroblasts, and their relationship to a subsequent calcification was studied. Glutaraldehyde caused an immediate and sustained massive cytosolic Ca2+ increase that was dose dependent and a several-fold increase in Pi. Calcification of cells followed within a week. The earliest calcification was observed in blebs formed on glutaraldehyde-treated cells. Live control cells or cells fixed with glutaraldehyde in Ca2+-free solution did not calcify under the same conditions. Concomitant increases in Ca2+ and Pi in glutaraldehyde-treated cells appear to underlie the mechanism of calcification, and the presence of extracellular Ca2+ during glutaraldehyde fixation promotes calcification.

Figure 1.

Photometric data of [Ca²⁺]_i response to 0.2% to 0.6% GA treatment in CaGr-1-loaded porcine AV fibroblasts. GA caused an immediate increase in [Ca²⁺]_i followed by progressive increases in [Ca²⁺]_i in a dose-dependent manner.

Figure 2.

A montage of pseudocolored images of a chain of CaGr-1-loaded fibroblasts treated with 0.6% GA demonstrating increases in [Ca²⁺]_i. Images were captured at time zero and every 12 minutes. [Ca²⁺]_i increase is seen mainly in perinuclear areas. Pseudocolors ranging from blue to white were assigned to 0 to 255 gray scales.

Figure 3.

[P_i]_i in cells treated with 0.6% GA in HBSS2.5 (solid bars) and in HBSS-0 (gray bars). GA caused an increase in [P_i]_i within hours especially in cells fixed in the presence of [Ca²⁺]_o. [P_i]_i remained elevated for 24 hours and returned to the ground status in a week. Error bars indicate SEM.

Figure 4.

A: Depletions of Ca²⁺ from HBSS2.5 by cells fixed with 0.6% GA in HBSS2.5 (•) and in HBSS-0 (○). HBSS2.5 Ca²⁺ leveled off for a few days after an initial drop. A precipitous decline of Ca²⁺ occurred with cells previously fixed in HBSS2.5, whereas Ca²⁺ remained level with cells previously fixed in HBSS-0. B: A progressive depletion of P_i from HBSS2.5 was caused by cells fixed with 0.6% GA in HBSS2.5. Cells fixed in HBSS-0 and incubated in HBSS2.5 did not deplete P_i.

Figure 5.

A: Calcein fluorescence micrographs of live control cells incubated in MEM2.5 for a week. Nuclei are brighter than the cytoplasm. B: Calcein-stained cells fixed in 0.6% GA in HBSS2.5 for 24 hours and incubated in HBSS2.5 for a week. Calcified cells and blebs (arrow) emitted strong fluorescence. Nuclei of heavily calcified cells are usually spared from calcification (arrowhead). The pale, noncalcified cells in the background are due to automated camera exposure time. Magnification, ×300.

Figure 6.

A: Calcein fluorescence of cells treated with 0.6% GA and incubated in HBSS2.5 showed an inverse relationship with Ca²⁺ depletion. B: Regression analysis yielded a coefficient of 0.79. Elimination of the outlier on day 1 increased the coefficient to 0.99.

Figure 7.

Electron micrographs of suspended cells fixed in 0.6% GA for 24 hours and incubated in HBSS2.5 for a week. A: A cell with calcified blebs. No calcific deposits are seen in the cytoplasm. Magnification, ×7500. B: A closer view of the tip of a bleb indicated by the arrowhead in A. Needle-shaped apatite crystals abut against the inner surface of the membrane. Magnification, ×32,500. C: A calcific deposit in what appears to be a swollen mitochondrion in a bleb. Magnification, ×8300. Mitochondria outside of the bleb show matrix condensation but no apatite. Radiating needles are seen in a closer view of the deposit (inset, ×34,000). D: A heavily calcified cell with diffuse apatite deposition in the cytoplasm. The nucleus did not calcify. Magnification, ×7500. E: A higher magnification of the perinuclear area in D. Needle-shaped apatite crystals are apparent. Magnification, ×32,500. F: Electron diffraction of a calcified cell yielded the powder pattern consistent with hydroxyapatite. G: In occasional cells, the deposits were selectively seen in nuclei. No calcified blebs but a ruffled plasma membrane due to trypsinization were present. Magnification, ×8500. H: A closer view of an area in G demonstrates needle-shaped apatite in the nucleus. Magnification, ×25,000.