Ann Thorac Surg 2001;71:S408-S409
© 2001 The Society of Thoracic Surgeons
Basic research
Protein adsorption in glutaraldehyde-preserved bovine pericardium and porcine valve tissues
Ming Shen, MD, PhDa,
Sophie M. Carpentier, PhDa,
Michelle Cambillau, PhDa,
Lin Chen, MDa,
Bernard Martinet, BSa,
Alain Carpentier, MD, PhDa
a Laboratory for the Study of Cardiac Grafts and Prostheses, UPRES 264 Université Paris VI, Hôpital Européen Georges Pompidou, Paris, France
Address reprint requests to Dr Shen, Laboratoire d’Etude des Greffes et Prothèses Cardiaques, Hôpital Broussais, 96 rue Didot, 75014 Paris, France
e-mail: labo.legpc{at}brs.ap-hop-paris.fr
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract
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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.
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Introduction
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Glutaraldehyde-pretreated porcine valves have been used for heart valve replacement since 1968 [1]. Calcification of leaflets is the most frequent cause of clinical failure, resulting in valve regurgitation or stenosis. Numerous studies have shown that porcine valvular calcification is associated with the presence of 
-carboxyglutamic acid–containing proteins after implantation both in animals and in humans [2]. Previous studies from our laboratory showed that the process of porcine valve calcification was associated with the adsorption of proteins and phospholipids [3]. Carpentier-Edwards pericardial bioprostheses have been in clinical use since 1980 and have shown better performance than porcine valve bioprostheses [4].
The objective of the present study was to investigate whether there are differences in protein adsorption between bovine pericardial and porcine valvular tissues that could correlate with the differences observed clinically.
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Material and methods
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Tissue preparation and implantation
Porcine aortic valve leaflets and bovine pericardium samples were treated by the Carpentier technique using 0.625% glutaraldehyde and sterilant treatment. The glutaraldehyde solution was prepared using a standard 25% commercially available solution (Merck, Darmstadt, Germany) in 20 mmol/L phosphate buffer, pH 7.4 [1]. The sterilant treatment used Tween 80 [5].
Twenty 12-day-old Wistar rats (CERJ, Genest-Saint-Isle, France) were chosen for this study. They were anesthetized using 100% diethyl ether gas. Two pieces of bovine pericardium and two pieces of porcine valve leaflet were then implanted in subcutaneous pouches over the back of these animals [5]. These samples were explanted 1 month after implantation. All explanted tissues were washed with 0.9% NaCl and freeze-dried.
Total protein content analysis
To assess the protein content, the dry bioprosthetic tissues were cut and homogenized (Ultra-Turrax, T25 IKA Labortechnik, Staufen, Germany) in Tris buffer (Tris, 14 mmol/L; NaCl, 120 mmol/L; KCl, 3 mmol/L; pH 7.4) for 2 minutes at 4°C, followed by centrifugation at 12,000 g for 30 minutes at 4°C. Protein dosage was then performed according to the method of Lowry modified by Peterson, and results expressed in micrograms per milligram of dry tissue.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
The extracted proteins (50 µg) were processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresis was performed on 8% to 12% and 12% to 16% separating gels with a 4% stacking gel, according to the method of Laemmli [6]. Samples were dissolved in a sodium dodecyl sulfate buffer (2-mercapto-ethanol, 10 mmol/L; Tris buffer, 100 mmol/L, pH 6.8; 30% glycerol; 2% sodium dodecyl sulfate), heated at 100°C for 3 minutes, and electrophoresed overnight at a constant voltage (100 V at 4°C). Proteins separated on the gel were visualized using Amido Schwartz (2%) staining.
The quantitative analysis was performed by densitometry (Bio-Profil, Vilber Lourmat, Marne-La-Vall
e, France). Results were expressed as percent of the total protein content. The significance level was set at p less than 0.05.
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Results
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The total protein content showed a significantly higher protein concentration in porcine valve tissue than in pericardial tissue: 149 ± 22.6 µg/mg dry tissue versus 108 ± 12.7 µg/mg dry tissue (Fig 1).
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Fig 1. Total protein content showed significantly higher protein concentration in porcine valve tissue than in bovine pericardial tissue.
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Electrophoretic analysis showed that in the pericardial tissue, four protein bands (17, 16, 15.5, and 13.5 kDa) showed decreased concentration when compared with porcine valve tissue, whereas one band (11 kDa) showed increased concentration (Fig 2).
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Fig 2. In the bovine pericardial tissue, four protein bands showed decreased concentration when compared with porcine valve tissue, whereas one band showed increased concentration.
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Comment
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Pericardial bioprostheses preserved with glutaraldehyde have been available as second-generation prostheses for valve replacement surgical procedures. Recent long-term data on Carpentier-Edwards pericardial bioprosthesis offer excellent clinical results and durability, particularly in the aortic position and for patients older than 70 years of age [4]. In young patients and during pregnancy biologic valves deteriorate more rapidly because of increased calcium metabolism and increased serum osteocalcin levels. However, Salazar and colleagues [7] showed that pregnancy does not accelerate the rate of deterioration of bovine pericardial bioprostheses. Glasmacher and associates [8] showed that pericardial valves are less prone to calcification than porcine valves. In previous studies we also found that bovine pericardium was less prone to calcification than porcine valves in a subcutaneous rat implant model (unpublished data).
In the present study, we found both a quantitative and qualitative difference in the proteins adsorbed in these two different bioprosthetic tissues. The proteins adsorbed in the tissues have a small molecular weight. They could correspond to noncollagenous bone matrix proteins, which are known to favor calcification. Studies with currently available specific antibodies did not permit us to identify these proteins. The difference between the adsorption of proteins in pericardial and bioprosthetic tissue is probably related to the structure of these tissues; bovine pericardium being denser than porcine valve tissue prevents the penetration of proteins after implantation. On the other hand, the 11-kd protein found at higher concentration in bovine pericardium raises the question of this protein being a protein mitigating calcification. Again, the currently available antibodies did not permit the identification of this protein. This identification is in progress.
The adsorption proteins were significantly different between bovine pericardium and porcine valve tissues after implantation in rats. Some proteins were found at higher concentrations in porcine valves, whereas another was more concentrated in bovine pericardium. Studies are being undertaken to identify these proteins, which may lead to a better understanding of the mechanism involved in the process of calcification.
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Acknowledgments
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We thank Martine Rancic for providing technical help.
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References
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Abstract
Introduction
