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Heat treatment mitigates calcification of valvular bioprostheses
Heat Treatment Mitigates Calcification of Valvular Bioprostheses Sophie M. Carpentier, PhD, Lin Chen, MD, Ming Shen, MD, PhD, Paul Fornes, MD, PhD, Bernard Martinet, Lillian J. Quintero, MS, Thomas H. Witzel, MS, and Alain F. Carpentier, MD, PhD Laboratoire d’Etude des Greffes et Prothe`ses Cardiaques, Universite´ Paris VI, Hoˆpital Broussais, Paris, France
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 6 2.3 mg Ca/mg (n 5 8) versus 239.9 6 2.9 mg Ca/mg (n 5 8); bovine pericardium: 19.5 6 8 mg Ca/mg (n 5 6) versus 108 6 10.3 mg Ca/mg (n 5 8); and porcine valve bioprostheses: 0 versus 11. Conclusions. Heat treatment of glutaraldehyde-treated bioprostheses significantly mitigated calcification in both subcutaneous and mitral position.
B
ioprosthetic heart valves developed in our laboratory in 1968 have the advantage of a reduced thrombogenicity over mechanical valves [1]; however, calcification after several years of implantation in young adults and in children has restricted their use [2– 4]. Significant calcium mitigation has been obtained by additional treatment of the glutaraldehyde-fixed tissue with surfactant [5]. In this article, we report another technique of calcium mitigation using heat treatment of the tissue in the glutaraldehyde solution. Our hypothesis was that heat may denature the proteins and phospholipids involved in the process of calcification. Preliminary results were reported in an earlier publication [6]. In this article we report further studies carried out to optimize heat treatment.
MgCl2 z 6H2O 4 g, NaCl 4.9 g, and HEPES 4.76 g in 1 L of distilled water. The pH was adjusted at 7.4 with 1 N NaOH. Bovine pericardial tissue and porcine aortic valves were obtained at slaughter. They were immediately placed in the 0.6% glutaraldehyde solution. The tissues were either cut into 0.5-cm2 pieces for subcutaneous implantation in rats or mounted into a frame for implantation in the mitral position in sheep. After 15 days of fixation in the glutaraldehyde solution, heat treatment was achieved by heating the solution containing the tissue, for periods of 15, 30, 45, and 60 days, with 20 leaflets/100 mL solution. Heating was obtained by placing glass culture flasks in an orbital shaking incubator (Rosi 1000, Barnstead/Thermolyse Corp, Dubuque, IA) at 70 rpm.
Material and Methods
Tissue Subcutaneous Implantation and Retrieval
Tissue Preparation Bovine pericardium or porcine valves were prepared according to our original method of glutaraldehyde pretreatment of heterologous tissues [1], with the sole difference that HEPES buffer was used instead of phosphate [5]. The glutaraldehyde solution was prepared from a 25% commercially available solution (Merck, Darmstadt, Germany) as follows: glutaraldehyde solution 26 mL, Presented at the VII International Symposium on Cardiac Bioprostheses, Barcelona, Spain, June 13–15, 1997. Address reprint requests to Dr S. Carpentier, Laboratoire d’Etude des Greffes et Prothe`ses Cardiaques, Hoˆpital Broussais, 96 rue Didot, 75014 Paris, France.
© 1998 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1998;66:S264 – 6) © 1998 by The Society of Thoracic Surgeons
Two-week-old (30 to 40 g) male and female Wistar rats were fed by their mother for 7 weeks and then given a normal animal laboratory diet. Before cusp implantation, the rats were anesthetized by diethyl ether and four pieces of aortic cusp were placed in subcutaneous pouches dissected in the dorsal wall. Two different treatments (two pieces each) were investigated in each animal and 10 rats were used for each treatment, so that 20 samples of implanted tissue were available for each treatment. After various periods (1 to 4 months), the rats were killed by an overdose of diethyl ether and the samples were retrieved. In addition, four nonimplanted samples per treatment were lyophilized for determination of initial calcium content. 0003-4975/98/$19.00 PII S0003-4975(98)01101-1
Ann Thorac Surg 1998;66:S264 – 6
Fig 1. Glutaraldehyde-preserved bovine pericardium heat treated at 20°, 42°, and 50°C for 2 months and then implanted for 3 and 4 months in newborn rats. Calcium mitigation is significant at 50°C. (The slight increase in calcium content between 3 and 4 months of implantation is not significant.)
Mitral Valve Implantation and Retrieval Technique in Sheep Nine heat-treated (50°C for 2 months) porcine valves and 7 control valve bioprostheses were implanted in 3-month-old sheep (25 to 30 kg). All animals received care in compliance with the Animal Welfare Act (PL 99-198). Mitral valve replacement (size, 25 mm) was performed through a left lateral thoracotomy and under extracorporeal circulation. Each animal received parenteral antibiotic therapy for 5 days postoperatively and was maintained on oral warfarin sodium for 6 weeks. Sacrifice of the surviving animals was performed after 5 months.
Analysis of Treated Tissues Before implantation, the following tissue analyses were performed: temperature shrinkage, NH2, histologic examination. For valves implanted in the mitral position, hemodynamic tests of frame-mounted porcine or pericardial valves were performed to assess valve durability and transvalvular gradients. The accelerated wear tester allowed the testing of valves at a rate of 1,000 cycles/min with a peak closing pressure of 120 mm Hg. After implantation, the implanted tissues were studied by x-ray analysis, spectrophotometry, and histologic examination (von Kossa’s technique).
CARDIAC BIOPROSTHESES CARPENTIER ET AL HEAT-TREATED GLUTARALDEHYDE BIOPROSTHESES
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Fig 2. Porcine valves heat treated at 50°C for 15 days (15d), 1 month (1m), and 2 months (2m) and then implanted for 2 and 3 months in newborn rats. Calcium mitigation is significant after a 2-month treatment at 50°C.
Duration of Heat Treatment Heat treatment at 50°C of porcine valves was investigated for periods between 15 days and 2 months. Calcium mitigation took place only after 2 months of treatment: 6.7 6 2.3 mg Ca/mg dry weight in the heat-treated group versus 245.4 6 4 mg Ca/mg dry weight in the control group after 3 months in rats (Fig 2). No mitigation was obtained with a heat treatment of 15 days or 1 month. Similar results were observed with pericardial tissues heat treated at 50°C for periods of 15 days to 3 months (19.5 6 8 mg Ca/mg dry weight in the heat-treated group versus 108 6 10.3 mg Ca/mg dry weight in the control group after 3 months in rats) (Fig 3). A longer duration of heat treatment did not mitigate calcification further.
Mitral Valve Implantation in Sheep Of the 16 sheep operated on, all survived the operation. They were sacrificed 20 weeks after implantation. Figure 4 shows the results of the x-ray analysis of the retrieved cusps.
Results Influence of Temperature Heat treatment at 50°C for 2 months markedly inhibited calcification of pericardial tissues subcutaneously implanted for 3 and 4 months. No significant calcium mitigation was observed in tissues stored at 20° or 42°C (Fig 1). Heat treatment at 60° and 70°C for 2 months did mitigate calcification but produced a thickening of the tissue leading to high transvalvular gradients. Heat treatment at 50°C for 2 months also inhibited calcification of porcine valves (Fig 2).
Fig 3. Bovine pericardium treated at 50°C for 15 days (15d), 1 (1m), 1.5 (1.5m), 2 (2m), and 3 months (3m) and then implanted for 3 and 4 months in newborn rats. Calcium mitigation is significant after a 2-month treatment at 50°C.
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CARDIAC BIOPROSTHESES CARPENTIER ET AL HEAT-TREATED GLUTARALDEHYDE BIOPROSTHESES
Fig 4. Heat-treated and control porcine valve bioprostheses in the mitral position in 16 juvenile sheep. The degree of calcification at retrieval 20 weeks after implantation was assessed by x-ray analysis. (0 5 none; 1 5 moderate; 11 5 important.)
Physical and Chemical Analysis Table 1 summarizes physical and chemical characteristics of heat-treated versus non– heat-treated tissues. There was a slight decrease in NH2 residues and no difference in temperature shrinkage and durability between the two groups. Histologic examination of heattreated tissues before implantation showed little change, namely a less dense spongiosa and fibrosa caused by some leaching of the proteins (Fig 5). The nonfibrillar and fibrillar matrix appeared looser than in the control group. Elastic fibers were normal.
Comment Since the introduction of glutaraldehyde in 1968 [1], numerous efforts have been made to minimize calcification of valvular bioprostheses. Pretreatment of bioprosthetic valve leaflets with magnesium chloride [5, 7], HEPES buffer [5], and detergent compounds [5, 7, 8] have been shown to mitigate calcification, but tissue calcification still exists in the long-term, particularly in the young adult population. The use of heat treatment is based on the hypothesis that heat may facilitate extraction and some denaturation of the phospholipids and proteins involved in the process of calcification. The role of extraction is suggested by the residue found after 2 months in the heated solution. Unfortunately the analysis of the residue did not permit us to identify specific components, most probably because of the glutaraldehyde fixation. The use of infrared spectroscopy may Table 1. Physical and Chemical Characteristics of Glutaraldehyde-Treated Tissues Porcine Valves
Bovine Pericardium
Characteristic
Control
50°C
Control
50°C
TS (°C) NH2a Durability (Mc)*
85 6 0.1 6.3 6 1.1 190
84 6 0.1 3.7 6 0.4 190
85 6 0.2 2.5 6 0.4 190
84 6 0.1 2.0 6 0.3 190
a
mole NH2/mole collagen.
* in million cycles.
Ann Thorac Surg 1998;66:S264 – 6
Fig 5. The histologic picture of a heat-treated glutaraldehyde-preserved porcine valve displays normal structure with slightly less dense tissue (arrow). (Orcein stain, 3100.)
prove to be useful in this regard. When compared with our previous methods of calcium mitigation [5, 6] heat treatment appears to achieve a superior efficacy. In summary the major findings of this study are as follows. Heat treatment of glutaraldehyde-preserved tissues does mitigate calcification under specific conditions of temperature and duration of treatment. The optimal temperature is 50°C, and the optimal duration of treatment is 2 months. Heat treatment does not modify significantly the tissue’s physical characteristics, although a slight increase in stiffness was noted without an increase in transvalvular gradient. As with other calcium mitigation methods, heat treatment does not fully prevent calcium phosphate deposition in the tissue, but it seems to be more efficient than any of the techniques previously described.
References 1. Carpentier A, Lemaigre G, Robert L, Carpentier S, Dubost C. Biological factors affecting long-term results of valvular heterografts. J Thorac Cardiovasc Surg 1969;58:467– 83. 2. Carpentier A, Deloche A, Relland J, et al. Six-year follow-up of glutaraldehyde preserved heterografts. J Thorac Cardiovasc Surg 1974;68:771– 82. 3. Magilligan DJ, Lewis JW, Stein P, Alam M. The porcine bioprosthetic heart valve: experience at 15 years. Ann Thorac Surg 1987;44:566–7. 4. Fiddler GI, Gerlis LM, Walker DR, Scott O, Williams GJ. Calcification of glutaraldehyde-preserved porcine and bovine xenograft valves in young children. Ann Thorac Surg 1983;35: 257– 61. 5. Carpentier A, Nashef A, Carpentier S, Goussef N, Ahmed A, Jones M. Techniques for prevention of calcification of valvular bioprostheses. Circulation 1984;70(Suppl 1):I-165– 8. 6. Carpentier SM, Carpentier AF, Chen L, Shen M, Quintero LJ, Witzel TH. Calcium mitigation in bioprosthetic tissues by iron pretreatment: the challenge of iron leaching. Ann Thorac Surg 1995;60:S332– 8. 7. Neethling WML, Meyer JM, Duyvene´ LJ. The effects of potassium phosphate, magnesium chloride and T6 as blocking reagents on tissue valve calcification. J Cardiovasc Surg 1989;30:317–21. 8. Jones M, Eidbo EE, Hilbert SL, Ferrans VJ, Clark RE. The effects of anticalcification treatments on bioprosthetic heart valves implanted in sheep. Trans Am Soc Artif Intern Organs 1988;34:1027–30.
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 6 2.3 mg Ca/mg (n 5 8) versus 239.9 6 2.9 mg Ca/mg (n 5 8); bovine pericardium: 19.5 6 8 mg Ca/mg (n 5 6) versus 108 6 10.3 mg Ca/mg (n 5 8); and porcine valve bioprostheses: 0 versus 11. Conclusions. Heat treatment of glutaraldehyde-treated bioprostheses significantly mitigated calcification in both subcutaneous and mitral position.
B
ioprosthetic heart valves developed in our laboratory in 1968 have the advantage of a reduced thrombogenicity over mechanical valves [1]; however, calcification after several years of implantation in young adults and in children has restricted their use [2– 4]. Significant calcium mitigation has been obtained by additional treatment of the glutaraldehyde-fixed tissue with surfactant [5]. In this article, we report another technique of calcium mitigation using heat treatment of the tissue in the glutaraldehyde solution. Our hypothesis was that heat may denature the proteins and phospholipids involved in the process of calcification. Preliminary results were reported in an earlier publication [6]. In this article we report further studies carried out to optimize heat treatment.
MgCl2 z 6H2O 4 g, NaCl 4.9 g, and HEPES 4.76 g in 1 L of distilled water. The pH was adjusted at 7.4 with 1 N NaOH. Bovine pericardial tissue and porcine aortic valves were obtained at slaughter. They were immediately placed in the 0.6% glutaraldehyde solution. The tissues were either cut into 0.5-cm2 pieces for subcutaneous implantation in rats or mounted into a frame for implantation in the mitral position in sheep. After 15 days of fixation in the glutaraldehyde solution, heat treatment was achieved by heating the solution containing the tissue, for periods of 15, 30, 45, and 60 days, with 20 leaflets/100 mL solution. Heating was obtained by placing glass culture flasks in an orbital shaking incubator (Rosi 1000, Barnstead/Thermolyse Corp, Dubuque, IA) at 70 rpm.
Material and Methods
Tissue Subcutaneous Implantation and Retrieval
Tissue Preparation Bovine pericardium or porcine valves were prepared according to our original method of glutaraldehyde pretreatment of heterologous tissues [1], with the sole difference that HEPES buffer was used instead of phosphate [5]. The glutaraldehyde solution was prepared from a 25% commercially available solution (Merck, Darmstadt, Germany) as follows: glutaraldehyde solution 26 mL, Presented at the VII International Symposium on Cardiac Bioprostheses, Barcelona, Spain, June 13–15, 1997. Address reprint requests to Dr S. Carpentier, Laboratoire d’Etude des Greffes et Prothe`ses Cardiaques, Hoˆpital Broussais, 96 rue Didot, 75014 Paris, France.
© 1998 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1998;66:S264 – 6) © 1998 by The Society of Thoracic Surgeons
Two-week-old (30 to 40 g) male and female Wistar rats were fed by their mother for 7 weeks and then given a normal animal laboratory diet. Before cusp implantation, the rats were anesthetized by diethyl ether and four pieces of aortic cusp were placed in subcutaneous pouches dissected in the dorsal wall. Two different treatments (two pieces each) were investigated in each animal and 10 rats were used for each treatment, so that 20 samples of implanted tissue were available for each treatment. After various periods (1 to 4 months), the rats were killed by an overdose of diethyl ether and the samples were retrieved. In addition, four nonimplanted samples per treatment were lyophilized for determination of initial calcium content. 0003-4975/98/$19.00 PII S0003-4975(98)01101-1
Ann Thorac Surg 1998;66:S264 – 6
Fig 1. Glutaraldehyde-preserved bovine pericardium heat treated at 20°, 42°, and 50°C for 2 months and then implanted for 3 and 4 months in newborn rats. Calcium mitigation is significant at 50°C. (The slight increase in calcium content between 3 and 4 months of implantation is not significant.)
Mitral Valve Implantation and Retrieval Technique in Sheep Nine heat-treated (50°C for 2 months) porcine valves and 7 control valve bioprostheses were implanted in 3-month-old sheep (25 to 30 kg). All animals received care in compliance with the Animal Welfare Act (PL 99-198). Mitral valve replacement (size, 25 mm) was performed through a left lateral thoracotomy and under extracorporeal circulation. Each animal received parenteral antibiotic therapy for 5 days postoperatively and was maintained on oral warfarin sodium for 6 weeks. Sacrifice of the surviving animals was performed after 5 months.
Analysis of Treated Tissues Before implantation, the following tissue analyses were performed: temperature shrinkage, NH2, histologic examination. For valves implanted in the mitral position, hemodynamic tests of frame-mounted porcine or pericardial valves were performed to assess valve durability and transvalvular gradients. The accelerated wear tester allowed the testing of valves at a rate of 1,000 cycles/min with a peak closing pressure of 120 mm Hg. After implantation, the implanted tissues were studied by x-ray analysis, spectrophotometry, and histologic examination (von Kossa’s technique).
CARDIAC BIOPROSTHESES CARPENTIER ET AL HEAT-TREATED GLUTARALDEHYDE BIOPROSTHESES
S265
Fig 2. Porcine valves heat treated at 50°C for 15 days (15d), 1 month (1m), and 2 months (2m) and then implanted for 2 and 3 months in newborn rats. Calcium mitigation is significant after a 2-month treatment at 50°C.
Duration of Heat Treatment Heat treatment at 50°C of porcine valves was investigated for periods between 15 days and 2 months. Calcium mitigation took place only after 2 months of treatment: 6.7 6 2.3 mg Ca/mg dry weight in the heat-treated group versus 245.4 6 4 mg Ca/mg dry weight in the control group after 3 months in rats (Fig 2). No mitigation was obtained with a heat treatment of 15 days or 1 month. Similar results were observed with pericardial tissues heat treated at 50°C for periods of 15 days to 3 months (19.5 6 8 mg Ca/mg dry weight in the heat-treated group versus 108 6 10.3 mg Ca/mg dry weight in the control group after 3 months in rats) (Fig 3). A longer duration of heat treatment did not mitigate calcification further.
Mitral Valve Implantation in Sheep Of the 16 sheep operated on, all survived the operation. They were sacrificed 20 weeks after implantation. Figure 4 shows the results of the x-ray analysis of the retrieved cusps.
Results Influence of Temperature Heat treatment at 50°C for 2 months markedly inhibited calcification of pericardial tissues subcutaneously implanted for 3 and 4 months. No significant calcium mitigation was observed in tissues stored at 20° or 42°C (Fig 1). Heat treatment at 60° and 70°C for 2 months did mitigate calcification but produced a thickening of the tissue leading to high transvalvular gradients. Heat treatment at 50°C for 2 months also inhibited calcification of porcine valves (Fig 2).
Fig 3. Bovine pericardium treated at 50°C for 15 days (15d), 1 (1m), 1.5 (1.5m), 2 (2m), and 3 months (3m) and then implanted for 3 and 4 months in newborn rats. Calcium mitigation is significant after a 2-month treatment at 50°C.
S266
CARDIAC BIOPROSTHESES CARPENTIER ET AL HEAT-TREATED GLUTARALDEHYDE BIOPROSTHESES
Fig 4. Heat-treated and control porcine valve bioprostheses in the mitral position in 16 juvenile sheep. The degree of calcification at retrieval 20 weeks after implantation was assessed by x-ray analysis. (0 5 none; 1 5 moderate; 11 5 important.)
Physical and Chemical Analysis Table 1 summarizes physical and chemical characteristics of heat-treated versus non– heat-treated tissues. There was a slight decrease in NH2 residues and no difference in temperature shrinkage and durability between the two groups. Histologic examination of heattreated tissues before implantation showed little change, namely a less dense spongiosa and fibrosa caused by some leaching of the proteins (Fig 5). The nonfibrillar and fibrillar matrix appeared looser than in the control group. Elastic fibers were normal.
Comment Since the introduction of glutaraldehyde in 1968 [1], numerous efforts have been made to minimize calcification of valvular bioprostheses. Pretreatment of bioprosthetic valve leaflets with magnesium chloride [5, 7], HEPES buffer [5], and detergent compounds [5, 7, 8] have been shown to mitigate calcification, but tissue calcification still exists in the long-term, particularly in the young adult population. The use of heat treatment is based on the hypothesis that heat may facilitate extraction and some denaturation of the phospholipids and proteins involved in the process of calcification. The role of extraction is suggested by the residue found after 2 months in the heated solution. Unfortunately the analysis of the residue did not permit us to identify specific components, most probably because of the glutaraldehyde fixation. The use of infrared spectroscopy may Table 1. Physical and Chemical Characteristics of Glutaraldehyde-Treated Tissues Porcine Valves
Bovine Pericardium
Characteristic
Control
50°C
Control
50°C
TS (°C) NH2a Durability (Mc)*
85 6 0.1 6.3 6 1.1 190
84 6 0.1 3.7 6 0.4 190
85 6 0.2 2.5 6 0.4 190
84 6 0.1 2.0 6 0.3 190
a
mole NH2/mole collagen.
* in million cycles.
Ann Thorac Surg 1998;66:S264 – 6
Fig 5. The histologic picture of a heat-treated glutaraldehyde-preserved porcine valve displays normal structure with slightly less dense tissue (arrow). (Orcein stain, 3100.)
prove to be useful in this regard. When compared with our previous methods of calcium mitigation [5, 6] heat treatment appears to achieve a superior efficacy. In summary the major findings of this study are as follows. Heat treatment of glutaraldehyde-preserved tissues does mitigate calcification under specific conditions of temperature and duration of treatment. The optimal temperature is 50°C, and the optimal duration of treatment is 2 months. Heat treatment does not modify significantly the tissue’s physical characteristics, although a slight increase in stiffness was noted without an increase in transvalvular gradient. As with other calcium mitigation methods, heat treatment does not fully prevent calcium phosphate deposition in the tissue, but it seems to be more efficient than any of the techniques previously described.
References 1. Carpentier A, Lemaigre G, Robert L, Carpentier S, Dubost C. Biological factors affecting long-term results of valvular heterografts. J Thorac Cardiovasc Surg 1969;58:467– 83. 2. Carpentier A, Deloche A, Relland J, et al. Six-year follow-up of glutaraldehyde preserved heterografts. J Thorac Cardiovasc Surg 1974;68:771– 82. 3. Magilligan DJ, Lewis JW, Stein P, Alam M. The porcine bioprosthetic heart valve: experience at 15 years. Ann Thorac Surg 1987;44:566–7. 4. Fiddler GI, Gerlis LM, Walker DR, Scott O, Williams GJ. Calcification of glutaraldehyde-preserved porcine and bovine xenograft valves in young children. Ann Thorac Surg 1983;35: 257– 61. 5. Carpentier A, Nashef A, Carpentier S, Goussef N, Ahmed A, Jones M. Techniques for prevention of calcification of valvular bioprostheses. Circulation 1984;70(Suppl 1):I-165– 8. 6. Carpentier SM, Carpentier AF, Chen L, Shen M, Quintero LJ, Witzel TH. Calcium mitigation in bioprosthetic tissues by iron pretreatment: the challenge of iron leaching. Ann Thorac Surg 1995;60:S332– 8. 7. Neethling WML, Meyer JM, Duyvene´ LJ. The effects of potassium phosphate, magnesium chloride and T6 as blocking reagents on tissue valve calcification. J Cardiovasc Surg 1989;30:317–21. 8. Jones M, Eidbo EE, Hilbert SL, Ferrans VJ, Clark RE. The effects of anticalcification treatments on bioprosthetic heart valves implanted in sheep. Trans Am Soc Artif Intern Organs 1988;34:1027–30.