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Background: Although glutaraldehyde fixation is known to reduce immunogenicity and degeneration of heart valve bioprostheses, some degree of immunogenicity persists, which may trigger calcification. The aims of this study were to: (1) define the role of alpha-1,3-galactosyltransferase (alpha-Gal) antigen in valve calcification by comparing alpha-Gal-positive and alpha-Gal-deficient (GT-KO) pig pericardium; and (2) elucidate the role of human anti-Gal antibodies in the process of calcification and to determine the potential influence of different tissue-fixation techniques., Methods: Glutaraldehyde-treated pericardium from alpha-Gal-positive and GT-KO pigs, with or without pre-labeling with human anti-Gal antibodies, were implanted in rats during 1 month., Results: In glutaraldehyde-fixed pericardium, calcification levels were significantly lower in GT-KO pig pericardium (132.8 +/- 5.8 microg/mg) as compared with alpha-Gal-positive pig pericardium (155.7 +/- 7.1 microg/mg) (p < 0.015). In glutaraldehyde-fixed pig pericardium followed by a mix of formaldehyde, ethanol and Tween 80 (FET), the calcification levels were lower in GT-KO pig pericardium (0.35 +/- 0.1 microg/mg) as compared with alpha-Gal-positive pig pericardium (4.6 +/- 4.2 microg/mg). In glutaraldehyde-fixed pig pericardium + FET pre-incubated with human anti-Gal antibodies, calcification levels were significantly greater in alpha-Gal-positive pig pericardium (43.8 +/- 8.5 microg/mg) as compared with GT-KO pig pericardium (5.7 +/- 2.9 microg/mg) (p < 0.0001)., Conclusions: This study demonstrates the role of alpha-Gal antigen and human alpha-Gal antibodies in the calcification process of valvular bioprostheses. It is suggested that GT-KO pig pericardium could be beneficial as a new source of material for heart valve bioprostheses., (Copyright (c) 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

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.

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 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.

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.

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.

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

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.

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.

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreated porcine valves after implantation in humans. Thirty-eight glutaraldehyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-binding proteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted values, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted values and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these values are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.

Background and Aim of the Study: The mechanism of valvular bioprostheses calcification is still unknown, but early studies showed increased Gla-protein content in calcified valves. Using an experimental model, which reproduces the clinical process, we therefore analyzed the role of minerals and proteins in bioprosthetic valvular calcification., Methods: Glutaraldehyde pretreated porcine valves were studied before and after implantation in rats by X-ray, calcium (Ca) and phosphorus (P) measurement, Fourier Transform Infrared (FTIR) spectroscopy, SDS-PAGE and 45Ca ligand blotting of the extracted proteins., Results: Before implantation, there was no X-ray calcification with very little Ca and P content. After implantation, X-ray calcifications appeared on day seven with increased Ca and P up to day 35 (p < 0.05, ANOVA). FTIR revealed structural proteins alone before implantation, plus minor proportions of lipids on day two, which always preceded Ca and P appearance. Ca and P increased up to day 35, first as amorphous and changed in carbapatite over time. SDS-PAGE before implantation revealed two proteins (66-kD and 54-kD) alone, which were sustained up to day 35. The 66-kD had 45Ca affinity. On day two, many other proteins appeared on SDS-PAGE, four of which (52, 45, 14 and below 14-kD) with 45Ca affinity. Protein pattern did not change from day two to 35., Conclusions: Valvular bioprosthesis calcification is associated with progressive increase in Ca and P content and at least five calcium-binding proteins: one intrinsic valvular protein, pre-existing to implantation, plus four other, extrinsic valvular proteins adsorbed within the tissue after implantation.

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.

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.

Calcium and gla-protein content are increased in the calcifications of cardiac bioprostheses. Such calcifications are more frequent during growth, pregnancy and renal failure when bone gla-protein levels are elevated. We investigated whether bone gla-protein and other markers of calcium metabolism play a role in bioprostheses calcifications. Forty-seven patients were separated into 2 groups according to the presence (group A, n = 9) or absence (group B, n = 38) of bioprostheses calcifications, as assessed by echo-doppler and surgery. Plasma levels of calcium, phosphorus, magnesium, creatinine and alkaline phosphatases were measured by standard laboratory methods, parathormone and those of bone gla-protein by specific radioimmunoassays. Results (mean +/- SEM) were compared (group A versus group B, P < 0.01) using Student's test and one-factor variance analysis (ANOVA). Age was similar in both group (53 +/- 12.9 vs 50 +/- 12.3 yrs), whereas duration of implant was greater in group A (104 +/- 12.4 vs 66 +/- 6.5 months, P < 0.01). No statistically significant difference was found between group A and B concerning biochemical and/or hormonal markers of calcium metabolism. These negative results merit to be discussed, and further studies will be needed to explore the potential role of circulating bone gla-protein in bioprostheses calcifications.

Implantation of a valvular bioprosthesis is one of the best treatments for valvular disease, particularly in children. Unfortunately, their use is limited as calcifications develop over time. Prevention of these calcifications is still an unresolved problem that is under study by Professor Carpentier and coworkers. These investigators have developed an experimental model in which accelerated development of calcifications occurs: fragments of porcine valves implanted under the skin of rats become calcified in a few days. Prior to implantation, the fragments are pretreated in an attempt to delay or prevent the development of calcifications. Two, four, six and eight months after implantation, the fragments are removed and calcifications are looked for using microradiography. Two hundred fragments have been studied. The material and technique used in our study are described. Results are semi-quantitative, based on the volume of calcifications. We conclude that microradiography is faster and more reliable than histology (the only technique used up till now) as it allows to study the entire fragments.

Calcification of valvular bioprostheses in children is a major problem that has stimulated extensive research in our laboratory. In previous reports, we have shown that the rate of calcification could be reduced by decreasing the phosphate content in the tissue or by blocking calcification binding sites with Mg++ and/or with a surfactant (Tween 80). Since then, we have systematically investigated incorporation of numerous other surfactants and of polyacrylamide within the tissue, and these investigations form the basis of this report. The methods of investigation included subcutaneous implantation of treated tissues in growing rabbits, stability tests, and intracardiac implantation in sheep. Results showed that surfactants differed in their efficacy in mitigating calcification, with N-lauroylsarcosine and triton X-100 being the most efficient. Polyacrylamide incorporation was also efficient in calcification mitigation, but this effect was lost after flexibility testing in vitro or implantation in vivo, a drawback that suggests further research is necessary into stabilization of this polymer. This report not only brings attention to new alternatives in calcification mitigation treatment of bioprosthetic tissues but also underlines important points of methodology.