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Background . Preliminary studies showed that heat treatment of glutaraldehyde preserved valvular bioprostheses mitigates calcification. This study was carried out to define the physicochemical characteristics of the heat-treated tissues to elucidate the mechanism involved in the mitigation. Methods . Glut bovine pericardium or porcine valve samples were treated at 50°C in a 0.625% glutaraldehyde solution for 2 months. Some samples underwent assay for shrinkage temperature, moisture content, ninhydrin test, and acid hydrolysis, and other samples were incubated in human serum for 3 days and then analyzed by electrophoresis to study protein adsorption. Results . Heat treatment mitigated calcification without adversely affecting shrinkage temperature (84.81°C versus 83.95°C) and moisture content (78.68% versus 78.71%). A significant reduction in free amino groups (0.15 versus 0.37 mol NH 2 /mol collagen) and a significant increase in resistance to acid hydrolysis were observed. Total protein content was similar, but significant differences were found for four proteins adsorbed in the tissues (167, 45, 11.6, and 10 kDa). Conclusions . The anticalcification effect of heat treatment may be attributed to structural changes, lipid extraction, increased resistance, and modifications of the type and concentration of the proteins adsorbed in the tissue.

Background . Several techniques have been proposed to mitigate calcification of glutaraldehyde-preserved bioprostheses. None has been fully satisfactory. Knowing that heat induces significant molecular changes, we investigated the potential benefit of the heat treatment of glutaraldehyde-fixed tissues. Methods . Samples of porcine valves and bovine pericardium treated in 0.625% glutaraldehyde were submitted to temperatures from 37° to 70°C for 2 to 12 weeks and then implanted subcutaneously in newborn rats for 3 months. In a second protocol, nine heat-treated porcine valve bioprostheses and seven control porcine valve bioprostheses were implanted in the mitral position in sheep for 20 weeks. Results . Spectrophotometry and x-ray analysis comparing heat-treated versus non–heat-treated tissues showed the following: porcine valve: 6.7 ± 2.3 μg Ca/mg (n = 8) versus 239.9 ± 2.9 μg Ca/mg (n = 8); bovine pericardium: 19.5 ± 8 μg Ca/mg (n = 6) versus 108 ± 10.3 μg Ca/mg (n = 8); and porcine valve bioprostheses: 0 versus ++. Conclusions . Heat treatment of glutaraldehyde-treated bioprostheses significantly mitigated calcification in both subcutaneous and mitral position.

Background . The methods currently used to analyze the process of calcification of bioprostheses give only global information on calcium deposition. We investigated the potential advantage of infrared spectroscopy, which makes it possible to analyze the various components of the calcification process, ie, lipids, proteins, and calcium deposits. Methods . Sixty porcine aortic leaflets were fixed in 0.6% glutaraldehyde and then subsequently implanted in 10-day-old Wistar rats. The valve leaflets were removed 2, 7, 14, 21, 35, and 56 days after implantation. Results . Before implantation infrared spectroscopic analysis revealed the presence of proteins only. On day 2 after implantation, all valves showed minor lipid deposits. On day 7, amorphous calcium phosphate was detected. Between days 7 and 14, crystalline forms of calcium phosphate appeared and amorphous calcium phosphate progressively changed into carbapatite over the 56-day period. Conclusions . Infrared spectroscopy yields valuable additional information on the nature and kinetics of the various components of glutaraldehyde-treated tissues after implantation. It may prove to be important in the evaluation of new techniques of calcium mitigation.

Background . The incidence of calcification of porcine valve bioprostheses shows important, and as yet unexplained, variations. Previous studies by others showed that osteopontin and osteocalcin are expressed in calcified porcine valve bioprostheses. However, no study has yet explored other proteins that could also be involved. Methods . Twelve porcine valve bioprostheses were retrieved from 12 patients and were separated into two groups. Group 1 (n = 6) had early calcification after 4 to 9 years (mean, 6 ± 2.3 years). The mean age of the patients at the time of implantation was 46 ± 9 years. Group 2 (n = 6) had no calcification after 8 to 14 years (mean, 12 ± 2.8 years). The mean age was 47 ± 13.4 years. These valves were analyzed by electrophore-sis, and the bands were quantified by densitometry. Results . A 14-kd protein showed a 50% increase in the calcified group. A 31-kd protein found in the calcified group was not detected in the noncalcified group. Three other proteins (45, 39, and 28 kd) showed reduced adsorption in the calcified group. Conclusions . Important differences were found in the proteins adsorbed in calcified and noncalcified bioprostheses after implantation in patients. Besides osteopontin and osteocalcin, several other proteins may play a role in the process of calcification of valvular bioprostheses.

Background . Proteins adsorbed by bioprosthetic tissues after implantation play a major role in the process of calcification. We investigated whether there are differences in protein adsorption between bovine pericardial and porcine valvular tissues that could correlate with the differences observed clinically. Methods . Glutaraldehyde-treated bovine pericardial and porcine valve samples were implanted subcutaneously in rats and retrieved 1 month after implantation. Total protein content was assessed by Lowry's method. Qualitative analysis was performed by polyacrylamide gel electrophoresis. Quantitative analysis was performed by densitometry. Results . Total protein content showed a higher protein concentration in porcine valve tissue than in pericardialtissue: 149 ± 22.6 μg/mg dry tissue versus 108 ± 12.7 μg/mg dry tissue (38% increase). In pericardial tissue, four protein bands (17, 16, 15.5, and 13.5 kd) showed decreased concentration when compared with porcine valve tissue, whereas one band (11 kd) showed increased concentration. Conclusions . Significant differences were found in protein content between bovine pericardial and porcine valve tissues. Correlations with clinical findings may lead to a better understanding of the mechanism involved in the process of calcification, particularly the role played by the structure of the tissues.