Asian Cardiovasc Thorac Ann 2007;15:413-417
© 2007 Asia Publishing EXchange Ltd
Stentless Valves Treated by the L-Hydro Process in the Aortic Position in Sheep
Paulo C Santos, MD,
Luís R Gerola, MD,
Ivan Casagrande, MD1,
Ênio Buffolo, PhD,
David T Cheung, PhD
Department of Cardiovascular Surgery, Escola Paulista de Medicina (UNIFESP), São Paulo
1 Research Center of Labcor, Belo Horizonte, Brasil
For reprint information contact: Paulo C Santos, MD, Tel: 55 34 3235 0167, Fax: 55 34 3235 0020, Email: paulocir{at}hotmail.com, Rua Bernardo Cupertino 704, Martins, Uberlândia, MG, Brazil 38400-444.
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ABSTRACT
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Calcification of glutaraldehyde-treated bioprosthetic heart valves is a major cause of long-term failure. We studied porcine aortic valves treated by the L-Hydro process and implanted into 14 juvenile sheep (group 1). Another 10 sheep were implanted with glutaraldehyde-treated porcine bioprostheses (group 2). The animals were sacrificed after 150 days and the explanted valves were analyzed for calcification. Hemodynamic measurements by echocardiography and angiography were carried out prior to sacrifice. Macroscopic analysis showed calcification and loss of mobility of the leaflets in all group 2 implants and in one group 1 implant. Light microscopy showed foci of calcification in all group 2 implants and in 3 valves from group 1. A significant reduction in the level of calcification was found in porcine bioprostheses treated by the L-Hydro process and implanted into the juvenile sheep model.
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INTRODUCTION
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Glutaraldehyde has been reported to cause some host immunogenic reactions to the xenogenic tissue, which leads to inflammation, thrombogenicity, calcification, and consequently decreased durability.1 Hence, preservation of the native structure and normal function of the tissue, without the use of toxic cross-linkers such as glutaraldehyde, is the major focus of current research.2 Several materials have been developed for this purpose.3 However, the results in terms of decreased calcification have been inconsistent. The objective of this study was to compare hemodynamic performance and levels of calcification of porcine stentless prosthetic heart valves treated by either glutaraldehyde or a non-aldehyde process and implanted in the aortic position in juvenile sheep.
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MATERIALS AND METHODS
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Fresh porcine aortic roots were obtained from a local abattoir and transported to the tissue processing facility at –8°C in phosphate-buffered saline at pH 7.4. Porcine aortic roots undergoing the L-Hydro process (Labcor/Philogenesis, Belo Horizonte, MG, Brazil) were stored in 50% ethanol prior to tissue treatment. The L-Hydro process consists of mild extraction of antigenic substances, masking of non-extracted antigens with polyethylene glycol (PEG), which is a highly water-soluble ethylene oxide derivative that produces a nonionic surfactant on the membrane surface, and mild chemical oxidation followed by incorporation of a nonsteroidal antiinfammatory agent and heparin. The treated tissues were sterilized with a hydrogen peroxide solution. Glutaraldehyde-treated porcine aortic roots were fixed in 0.4% glutaraldehyde solution for 8 weeks and sterilized in 0.6% glutaraldehyde with 20% ethanol at 55°C.
Twenty-four juvenile Santa Inês sheep (Ovis aries), 21 males and 3 females, were randomly divided into 2 groups and implanted with either L-Hydro-treated or glutaraldehyde-treated porcine aortic roots. Group 1 comprised 14 sheep (mean age, 5.59 ± 0.50 months; mean weight, 31.7 ± 2.5 kg) implanted with L-Hydro-treated stentless porcine bioprostheses. Group 2 comprised 10 sheep (mean age, 5.5 ± 0.55 months; mean weight, 31.4 ± 2.7 kg) implanted with glutaraldehyde-treated stentless porcine valves. All implants were in the aortic position with reattachment of the native coronary arteries by a previously published protocol.4 Animals were initially anesthetized with intravenous atropine 1 mg and propofol 8 mL, and given antibiotic prophylaxis and methylprednisolone 250 mg. During the operation, succinylcholine 100 mg and continuous inhalation of 1.0% to 1.5% halothane were used. A lateral thoracotomy was performed at the level of the 4th left intercostal space. Arterial cannulation was carried out in the descending aorta, with venous cannulation in the right atrium. Extracorporeal circulation was started at a flow rate of 50 to 70 mL·kg–1·min–1 with cooling until the esophageal temperature reached 28°C. Anterograde cardioplegia was delivered via the aortic root. Every 25 min, blood cardioplegic solution was infused into the coronary ostia (10 mL·kg–1). The aortic valve was completely removed along the initial portion of the root of the aorta and the individual coronary ostia. The stentless prosthetic aortic roots were implanted in the orthotopic position, with reattachment of the coronary arteries.
Five hours after the implant procedure, antibiotic therapy with intramuscular ampicillin 1 g and gentamicin 1 mL·kg–1 was started twice a day for 36 hours. If a temperature higher than 40.5°C was noted, antibiotic therapy was continued for 7 days. Blood samples were collected after 1 week to monitor renal, hematological, and biochemical function. The animals were kept under close observation for 10 to 14 days, then transferred to the animal department in the Labcor Research Center for long-term follow-up. The sheep were kept in a suitable environment with an adequate and balanced diet for their age. Blood samples were collected monthly to monitor renal, hematological, and biochemical function. The animals were sacrificed 150 days after implantation. Just prior to sacrifice, angiocardiographic and echocardiographic hemodynamic measurements were obtained. The implants were removed for histologic analysis to evaluate microcalcification using van Kossa stain. Other organs were sent for pathologic analysis.
Student’s t test, the Wald-Wolfowitz test, and the Mann-Whitney U test were the statistical methods used in this study.
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RESULTS
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There were 4 operative deaths in each group. In group 1 (L-Hydro), one animal died from uncontrollable bleeding from the proximal suture line, one had a myocardial infarction, and two presented with refractory hypoxia of unknown origin. In group 2, two animals had uncontrollable bleeding from the proximal suture line, and the other two did not recover from the procedure. There was no significant difference in mean ejection fraction: 76% ± 5.96% in group 1, 77.5% ± 3.14% in group 2; p = 0.5224. The mean transvalvular gradients were: 3.51 ± 2.38 mm Hg in group 1, 4.88 ± 2.40 mm Hg in group 2; p = 0.2928. Doppler echocardiograms of all implants indicated that the valves were functioning normally and there was no evidence of valvular insufficiency, endocarditis, or left ventricular dysfunction.
The 16 surviving animals (10 in group 1 and 6 in group 2) were followed-up for 150 days. In this period, there were 8 deaths (6 in group 1 and 2 in group 2). In group 1, one animal became paraplegic and was sacrificed on the 6th postoperative day, one died on the 31st day due to endocarditis and another on the 32nd day of unknown cause (possibly arrhythmia). Three sheep died at 128, 146, and 132 days post-implantation, due to enterotoxemia, endocarditis, and an unknown cause; these animals were included in the pathologic study. In group 2, two sheep died of cardiac insufficiency 6 and 32 days postoperatively. The valve of the first animal was normal and the explanted valve of the second animal was calcified. The other 4 sheep were sacrificed as scheduled. The animals underwent angiography and echocardiography for analysis of hemodynamic variables. The mean pulmonary capillary pressure was 10.02 mm Hg in group 1 and 8.62 mm Hg in group 2, with no significant difference between groups (Mann-Whitney U test: p = 0.665). The mean cardiac output was 5.22 L·min–1 in group 1 and 3.76 L·min–1 in group 2 (p = 0.11). Angiography did not reveal any problems with the reattached coronary arteries, with no signs of obstruction, torsion, or areas suggesting ischemia on ventriculography. There was no significant valvular backflow in any of the sheep. With the catheter positioned first in the left ventricle and then in the aortic root, it was possible to measure the respective pressures to calculate the transvalvular gradient. The mean gradient (left ventricle to aorta) was 23.50 mm Hg in group 1 and 30.50 mm Hg in group 2 (p = 0.56).
The presence of microcalcification was assessed as optical density in micrographs. Microcalcification was observed in 3 of the 7 sheep in group 1 and in all 6 of the group 2 animals (Figure 1
). This difference was significant ( p = 0.043). Visible macroscopic calcification (Figure 2) was observed in 1 of 7 animals analyzed in group 1 and in all 6 of the group 2 animals ( p = 0.020). The difference in degree of calcification between the two groups was significant (Wald-Wolfowitz nonparametric test: p < 0.05).
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Figure 1. Light microscopy of an explanted valve treated by the L-Hydro process. The arrow indicates a focus of calcification. von Kossa stain, original magnification x100.
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Heart, liver, kidneys, lungs, and brain of all animals were removed and submitted to necropsy. There were no important differences in the weight or size of these organs. In one sheep in group 2 that underwent elective sacrifice, there were signs of congestion in all organs, inferred by increased weight and edema of the visceral veins. Three sheep in group 1 and one in group 2 had prosthetic endocarditis; there appeared to be no significant difference between the groups.
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DISCUSSION
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Calcification of the leaflets of a bioprosthetic heart valve leads to immobilization, perforation, and destruction of the graft within 10 to 15 years. This requires re-operation for replacement. Fox and colleagues5 demonstrated that the process of calcification of bioprostheses seems to be unrelated to inflammation or specific immunologic activity, and markers of the systemic inflammatory response are increased due to various factors. The detection of antibodies in valvular tissue after failure is probably due to a reaction secondary to the valvular lesion and exposure of new antigenic material in the valve implant, prior anticalcification treatment, and graft structure.6 There is one hypothesis that calcification of bioprostheses occurs because there are no vital cells in the implanted tissue to maintain extracellular calcium levels and limit the increase of free aldehyde groups, and there are empty spaces that become filled with calcium phosphate.7 Calcification is the most common cause of degeneration of bioprostheses, occurring mainly in glutaraldehyde-treated tissues. Levy and colleagues8 demonstrated that pretreatment of tissues with an aldehyde reagent is a prerequisite for calcification, even in areas that are not exposed to mechanical stress. The use of glutaraldehyde for preservation of bioprostheses was first introduced by Carpentier and colleagues,9 who were also responsible for the first clinical use in 1974. The medium-term results have been satisfactory: after 5 years of observation, 77% of patients studied had preserved function of bioprostheses in the mitral position, 89% in the aortic position, and 96% in the tricuspid position.
To decrease the deleterious effects of glutaraldehyde, several agents have been developed. Three types of anticalcification treatment (Bi-Linx, AOA, and No-React) for leaflets of the aortic valve and tissue of the aortic root were compared in grafts implanted subcutaneously in rats.10 These methods had distinct limitations, resulting in eventual progression or initiation of the calcification process.10 The tissue treatment in this study utilizes PEG to coat tissue matrices. Polyethylene glycol is nontoxic and chemically inert, and when introduced into the circulation, it is completely cleared by the kidneys without being metabolized.11 Polyethylene glycol was shown to be effective in several in vitro studies. When added to the solution for myocardial preservation, it enhanced functional viability for a prolonged period (up to 24 hours), a time frame much better than that with conventional cardioplegic solutions alone.12 This is due to the osmotic action of PEG, which stabilizes the membrane, making it less permeable to extracellular solutes, and consequently preventing cellular edema.13 Polyethylene glycol also has an immunosuppressive property that reduces the antigenicity of transplants by its interaction with specific antigens. Some investigators suggested that the immunosuppressive properties of PEG are due to its ability to form reversible complexes with lipids and antigens on cell membranes. This changes or masks antigens on the cell surface in a manner analogous to that described for the specific chemical link with allergens. Polyethylene glycol interferes with the activation of macrophages, which consequently activates T helper cells, resulting in tolerance to donor antigens and reduced immunogenicity. In addition to PEG coating, the L-Hydro process extracts porcine antigens under very mild conditions that preserve the integrity of tissue fibers. The remaining antigens are masked by PEG, and platelet receptor sites for collagen are also modified or masked, causing reduced platelet density on the surface. Polyethylene glycol inhibits platelet adhesion and spreading, and reduces calcium deposition, while retaining bio-stability; this stability might be linked to properties of PEG whereby covalent binding to amino groups of proteins stabilizes the tertiary structure of the protein.14
In this study, we implanted porcine aortic stentless bioprosthetic heart valves treated by the L-Hydro process in the orthotopic position in juvenile sheep. Implanting these bioprostheses in the subcoronary aortic position is justified because this is a well-defined animal model close to the human. The environmental conditions of the implants, such as mechanical stress, interaction with elements of blood, and biochemistry are close to those in humans. The sheep has been used in several studies as a large experimental animal model. This animal is docile and easily handled postoperatively. Young sheep have a fast rate of development and growth, and physiopathologic changes are similar to those occurring during human development. The animals were followed up for 150 days. A 5-month period was chosen as suggested by other investigators, as it is an adequate period for calcification of bioprosthetic heart valves.15 The postoperative mortality rate due to infection was similar to that in reports by other investigators.16 In group 2, two sheep died of cardiac insufficiency during follow-up, corroborated by necropsy data indicating congestion in all organs. The prosthesis in both cases had decreased mobility with signs of stenosis, but without signs of calcification or endocarditis. Another possible cause of cardiac dysfunction might be overestimation of the size of prosthesis, which might lead to a degree of valve insufficiency. It was found that the non-aldehydic (L-Hydro) prosthesis had the same hemodynamic performance as the glutaraldehyde-treated prosthesis, characterized by the absence of significant regurgitation and by mean pulmonary capillary pressure of 10 mm Hg in group 1 and 8.62 mm Hg in group 2. We concluded that preservation by the L-Hydro method protects the integrity as well as the physical and functional properties of the graft.
Cineangiography and aortography on the day of elective sacrifice verified that there was good preservation of the coronary ostia. Aortography showed no significant degree of insufficiency of the prosthesis and no significant left ventricle-to-aorta gradient in both groups. Hemodynamic data provide further evidence of the functional performance of L-Hydro-treated stentless aortic bioprostheses implanted in the subcoronary position. Macroscopic analysis verified calcification in the explanted bioprostheses of both groups. However, the degree of calcification in group 2 was significantly greater than in group 1 where only one valve had macroscopically visible calcification. Light microscopy with van Kossa stain revealed a significantly higher level of microcalcification in group 2 implants. As the presence of endocarditis was not significantly different between the two groups, we concluded that endocarditis did not influence the results. The prostheses of group 2 were more susceptible to macroscopically and microscopically detectable calcification, and there were deposits of thrombotic material. The hemodynamic repercussions of these changes were evidenced in the loss of mobility of the explanted leaflets, especially in the 2 animals of group 2 that died days before the programmed elective sacrifice. These prostheses were stenotic with significantly reduced leaflet mobility. These findings are consistent with previous observations reported by Schoen and colleagues17 and Vicentelli and colleagues18 regarding the deleterious action of glutaraldehyde on heterologous valvular tissue. In macroscopic analyses of bioprostheses preserved with PEG, there was no thrombus. It has been suggested that resistance to thrombus enables the formation of new endothelium. The integrity of the new endothelium was characterized by its contact with the conjunctive matrix.
In a recent review, Schoen and Levy19 showed that the genesis of calcification is multifactorial and starts primarily inside devitalized cells, usually by glutaraldehyde treatment. They suggested that inducers and inhibitory factors that regulate pathological calcification are similar to those occurring in the mineralization of bone. Therefore, preventive strategies including calcification inhibitors in tissues fixed with glutaraldehyde, removal or modification of components of calcification, alterations in the preservation by glutaraldehyde, and the use of non-aldehydic agents have been proposed recently. However, there is no conclusive evidence that clinical implants have benefited by these strategies. This study, like others, utilized large animals as an experimental model for evaluation of new technologies for the preservation of tissues used as substitute cardiac valves. However, there are limitations, and it is difficult to reproduce the effect of human cardiac diseases in these animal models. In our study, implanting L-Hydro-treated stentless porcine bioprostheses in the aortic orthotopic position in juvenile sheep was adequate as an experimental model because the biomechanical impact on the valve is very similar to that in humans. Furthermore, we have presented evidence that L-Hydro treatment caused a reduction of calcification in the cusps of the bioprosthesis. These results support a clinical study to definitively validate the real benefit of this tissue process for a xenograft bioprosthetic heart valve.
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REFERENCES
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- Garcia Paez JM, Jorge-Herrero E, Carrera A, Millan I, Rocha A, Calero P, et al. Chemical treatment and tissue selection: factors that influence the mechanical behaviour of porcine pericardium. Biomaterials 2001;22:2759–67.[Medline]
- Cunanan CM, Cabiling CM, Dinh TT, Shen SH, Tran-Hata P, Rutledge JH 3rd, et al. Tissue characterization and calcification potential of commercial bioprosthetic heart valves. Ann Thorac Surg 2001;71:S417–21.[Medline]
- Rao KP, Shanthi C. Reduction of calcification by various treatments in cardiac valves. J Biomater Appl 1999;13:238–68.[Abstract/Free Full Text]
- Grehan JF, Casagrande I, Oliveira EL, Santos PC, Pessa CJ, Gerola LR, et al. A juvenile sheep model for the long-term evaluation of stentless bioprostheses implanted as aortic root replacements. J Heart Valve Dis 2001;10:505–12.[Medline]
- Fox CS, Guo CY, Larson MG, Vasan RS, Parise H, O’Donnell CJ, et al. Relations of inflammation and novel risk factors to valvular calcification. Am J Cardiol 2006;97:1502–5.[Medline]
- Flameng W, Meuris B, Yperman J, De Visscher G, Herijgers P, Verbeken E. Factors influencing calcification of cardiac bioprostheses in adolescent sheep. J Thorac Cardiovasc Surg 2006;132:89–98.[Abstract/Free Full Text]
- Vasudev SC, Chandy T, Sharma CP. The antithrombotic versus calcium antagonistic effects of polyethylene glycol grafted bovine pericardium. J Biomater Appl 1999;14:48–66.[Abstract/Free Full Text]
- Levy RJ, Schoen FJ, Howard SL. Mechanism of calcification of porcine bioprosthetic aortic valve cusps: role of T-lymphocytes. Am J Cardiol 1983;52:629–31.[Medline]
- Carpentier A, Deloche A, Relland J, Fabiani JN, Forman J, Camilleri JP, et al. Six-year follow-up of glutaraldehyde-preserved heterografts. With particular reference to the treatment of congenital valve malformations. J Thorac Cardiovasc Surg 1974;68:771–82.[Medline]
- Walther T, Falk V, Diegeler A, Rauch T, Weigl C, Gummert J, et al. Effectiveness of different anticalcification treatments for stentless aortic bioprostheses. Thorac Cardiovasc Surg 1999;47:23–5.[Medline]
- Bhayana JN, Tan ZT, Bergsland J, Balu D, Singh JK, Hoover EL. Beneficial effects of fluosol-polyethylene glycol cardioplegia on cold, preserved rabbit heart. Ann Thorac Surg 1997;63:459–64.[Abstract/Free Full Text]
- Wicomb WN, Perey R, Portnoy V, Collins GM. The role of reduced glutathione in heart preservation using a polyethylene glycol solution, Cardiosol. Transplantation 1992;54:181–2.[Medline]
- Collins GM, Wicomb WN, Levin BS, Verma S, Avery J, Hill JD. Heart preservation solution containing polyethyleneglycol: an immunosuppressive effect? Lancet 1991;338:890–1.[Medline]
- Vasudev SC, Chandy T. Polyethylene glycol-grafted bovine pericardium: a novel hybrid tissue resistant to calcification. J Mater Sci Mater Med 1999;10:121–8.[Medline]
- Herijgers P, Ozaki S, Verbeken E, Van Lommel A, Racz R, Zietkiewicz M, et al. Calcification characteristics of porcine stentless valves in juvenile sheep. Eur J Cardiothorac Surg 1999;15:134–42.[Abstract/Free Full Text]
- Gott JP, Girardot MN, Girardot JM, Hall JD, Whitlark JD, Horsley WS, et al. Refinement of the alpha aminooleic acid bioprosthetic valve anticalcification technique. Ann Thorac Surg 1997;64:50–8.[Abstract/Free Full Text]
- Schoen FJ, Hirsch D, Bianco RW, Levy RJ. Onset and progression of calcification in porcine aortic bioprosthetic valves implanted as orthotopic mitral valve replacements in juvenile sheep. J Thorac Cardiovasc Surg 1994;108:880–7.[Abstract/Free Full Text]
- Vincentelli A, Latremouille C, Zegdi R, Shen M, Lajos PS, Chachques JC, et al. Does glutaraldehyde induce calcification of bioprosthetic tissues? Ann Thorac Surg 1998;66(6 Suppl):S255–8.[Medline]
- Schoen FJ, Levy RJ. Calcification of tissue heart valve substitutes: progress toward understanding and prevention. Ann Thorac Surg 2005;79:1072–80.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
