Sophie Carpentier

Heat treatment mitigates calcification of valvular bioprostheses

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 degrees to 70 degrees 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 microg Ca/mg (n = 8) versus 239.9+/-2.9 /g Ca/mg (n = 8); bovine pericardium: 19.5+/-8 microg Ca/mg (n = 6) versus 108+/-10.3 microg 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.

Calcium Mitigation in Bioprosthetic Tissues by Iron Pretreatment: The Challenge of Iron Leaching

Preliminary studies in our laboratory have shown that iron pretreatment of glutaraldehyde-preserved tissues inhibited calcification. The present study was designed to further investigate this finding and to optimize the technique. Porcine valve tissue and bovine pericardium preserved in glutaraldehyde and pretreated by iron at different concentrations and incubation times were implanted either subcutaneously in rats or as a mitral valve substitute in sheep. Compared with control groups, calcification was markedly inhibited in the groups pretreated with Fe3+. There was a direct correlation between the degree of calcification and the iron content within the tissue. A minimal iron content within the tissue of 0.5% for porcine valve tissue and 0.2% for pericardial tissue was found to be necessary for calcium mitigation. A progressive leaching of Fe3+ was observed that could be stabilized by using a higher concentration of iron in the solution and or by additional pretreatment in glutaraldehyde at high temperature, but only for subcutaneously implanted tissues. By contrast, leaching was faster and more complete in the iron-pretreated tissue valves placed in the circulating blood environment, therefore resulting in calcified valves. Pretreatment in glutaraldehyde at high temperature (50 degrees C) alone was able to mitigate calcification in both subcutaneous and circulating blood environments, a new finding that may lead to an improved method for glutaraldehyde preservation of tissues.

Effect of human immunoglobulins on the immunogenicity of porcine bioprostheses

BACKGROUND Glutaraldehyde fixation (GT) is known to reduce immunologic reactions and tissue degeneration after implantation in humans. Sterilization after glutaraldehyde fixation (G-ST) improves the safety and reduces the tendency of GT valves to calcify. Intravenous immunoglobulins (IVIg) have been shown to reduce xenogeneic response against porcine tissue. We have investigated the effect of these fixation procedures combined with and without IVIg on the antigenicity of bioprostheses. METHODS Lewis adult rats were implanted subcutaneously with a fresh, GT, or G-ST porcine heart valve pre- or posttreated with different amounts of IVIg. We followed by enzyme-linked immunosorbent assay and IgM and IgG titers against protein extracts from the porcine heart valves after implantation. Cellular reactivity was assessed in xenogeneic lymphoendothelial coculture experiments. Calcification content was also examined. RESULTS Glutaraldehyde fixation partially decreased the humoral response against proteins of the implant but elicited a cellular xenogeneic response. Sterilization reduced these reactivities, but retained antigenicity. Intravenous immunoglobulin postincubated with GT valves before implantation reduced the antigenicity of the tissue to the same extent as G-ST valves, but had no effect on valvular tissue calcification. CONCLUSIONS Our studies demonstrate that IVIg or the sterilization procedure (ST) reduced the cellular response against glutaraldehyde-fixed valves (GT), whereas reduced calcification was observed only with ST.

Results of up to 9 years of high-temperature- fixed valvular bioprostheses in a young population

BACKGROUND Bioprosthetic valve replacement in young patients remains a controversial issue due to a high rate of early calcification. Previous studies in our laboratory have shown that high-temperature fixation of glutaraldehyde preserved bioprosthesis (HTF) mitigates calcification. The first clinical application of this technique was started in 1991. METHODS From January 1991 to September 1998, 50 patients in whom anticoagulants were contraindicated underwent single aortic valve replacement (n = 33) or mitral valve replacement (n = 17) using HTF bioprostheses. The age of the patients ranged from 7 months to 35 years (mean 22.7+/-6.8 years). The mean New York Heart Association status was 2.4. Mean follow-up 4 years +/- 1.8 for a total follow-up of 196 patient-years. RESULTS There were no operative deaths and but there were two late deaths, one valve related. Structural failure occured in 4 patients (2%/patient-year) requiring a reoperation in 3 patients (1.5%/patient-year). No endocarditis or thromboembolic episodes were observed. At late examination (June 2000), 46 patients (92%) were in New York Heart Association class I or II, with a well functioning valve. CONCLUSIONS Replacement with HTF bioprostheses in young patients has demonstrated encouraging midterm results with a low incidence of structural failure

Protein adsorption of calcified and noncalcified valvular bioprostheses after human implantation

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 electrophoresis, 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.

Infrared spectroscopy in the evaluation of the process of calcification of valvular bioprostheses

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.

Do donor or recipient species influence calcification of bioprosthetic tissues

To determine whether donor or recipient species influence calcification of bioprosthetic tissues, glutaraldehyde-treated valvar or pericardial specimens from different species (calf, sheep, pig) were subcutaneously implanted in different animals (rat, rabbit, cow, hen). Significant differences in the rate of calcification of the implanted specimens were found, which have important practical and theoretical implications for the development of valvular bioprostheses.

Biochemical properties of heat-treated valvular bioprostheses

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 degrees 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 degrees C versus 83.95 degrees C) and moisture content (78.68% versus 78.71%). A significant reduction in free amino groups (0.15 versus 0.37 mol NH2/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.

Protein adsorption in glutaraldehyde-preserved bovine pericardium and porcine valve tissues

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 Lowrys 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 pericardial tissue: 149+/-22.6 microg/mg dry tissue versus 108+/-12.7 microg/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.