Asian Cardiovasc Thorac Ann 2005;13:203-207
© 2005 Asia Publishing EXchange Ltd
Comparative Study of the L-Hydro Process and Glutaraldehyde Preservation
Vinicius JS Nina, PhD,
Pablo MA Pomerantzeff, PhD,
Ivan SJ Casagrande, MD1,
David T Cheung, PhD1,
Carlos MA Brandão, PhD,
Sérgio A Oliveira, PhD
The Heart Institute (HC-InCor), University of São Paulo Medical School, São Paulo, Brazil
1 The International Heart Institute of Montana, Missoula, MT, USA
For reprint information contact: Vinicius JS Nina, PhD Tel: 55 98 3227 9191 Fax: 55 98 3232 8700 Email: rvnina{at}aol.com, Rua Sebastião Archer, 101 Olho D’ Água, CEP: 65065-480, São Luís-MA, Brazil.
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ABSTRACT
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Commercial bioprosthetic heart valves are commonly preserved in glutaraldehyde and are cytotoxic to host cells, preventing spontaneous endothelialization. The aim of this study was to demonstrate the potential for in vivo endothelialization of bioprostheses treated by the L-Hydro process which consists of mild extraction of antigenic substances and incorporation of antiinflammatory and antithrombotic agents. Seven stented porcine heart valves treated by the L-Hydro process and 3 glutaraldehyde-fixed porcine heart valves were implanted in the mitral position in juvenile sheep. The valves were evaluated by echocardiography, angiography, histology, and histochemistry. No hemodynamic differences were observed, but scanning and transmission electron microscopy showed nearly complete coverage by endothelial cells of all leaflets in the L-Hydro-treated valves after 5 months of implantation. The endothelial cells were in direct contact with the underlying collagen and expressed von Willebrand-related antigens. The surfaces of the glutaraldehyde-treated valves were covered by fibrin, macrophages, calcium, and thrombotic material; only sparse endothelial cells were observed and contact with the underlying tissue was incomplete. These data indicate that L-Hydro-treated porcine valves are capable of inducing spontaneous endothelialization.
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INTRODUCTION
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Over the years, it has been suggested that the inability of cardiac bioprostheses to restore viable cardiac tissue may be the main cause of thrombogenicity, inflammation, and limited long-term durability.1 Previous reports have demonstrated that cellular extraction and removal of debris from inside the leaflets can substantially reduce antigenicity and the need for glutaraldehyde (GA) fixation.2–3 Moreover, maintenance of the integrity of the collagen matrix can be assured by recellularization of the leaflet with host cells.4–7 Therefore, preservation of the natural structure and function of valve tissue is currently the most important goal in the development of substitute devices for heart valves.7 However, complete spontaneous endothelialization of the valve leaflets has not yet been demonstrated with conventional tissue preservation methods. In this study, the potential for in vivo endothelialization of the porcine stented bioprosthesis was tested utilizing a new non-glutaraldehyde process of tissue preservation.
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MATERIALS AND METHODS
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Porcine valves were obtained from a slaughterhouse approved by the Brazilian Ministry of Agriculture and Sanitary Surveillance. After mounting in a stent, the valves were treated by the L-Hydro (Labcor/Philogenesis, Belo Horizonte, MG, Brazil) process which consists of 3 distinct steps. First, mild extraction of antigenic substances is combined with masking of un-removed antigens using polyethylene glycol, under controlled chemical oxidation and specific physical conditions that protect extracellular structures such as collagen and elastin. Second, a nonsteroidal antiinflammatory (aspirin-like) substance and an antithrombotic (heparin-like) agent are incorporated into the tissue. Third, the tissue is sterilized with an aqueous phase of hydrogen peroxide.
The animal experiments were performed with the approval of the Scientific and Ethics Committee of the School of Medicine of the University of São Paulo, and in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care.8 Thirteen Santa Inês sheep, with ages ranging from 4 to 6 months, and weighing between 25 and 35 kg, were divided into 2 groups: a test group of 9 animals implanted with an L-Hydro-treated porcine bioprosthesis; and a control group of 4 animals implanted with a 0.4% glutaraldehyde-fixed porcine bioprosthesis with no anti-calcification agent. Three animals (2 in the test group and 1 in the control group) died perioperatively (< 24 hours) and were excluded from the study. All deaths were non-valve related, being caused by intraoperative air embolism, ileus, and ventricular arrhythmia. The 25 mm valves were implanted in the mitral position through a left lateral thoracotomy in the 4th intercostal space, under ventricular fibrillation. The operations were concluded with drainage of the chest cavity and closure of the chest wall in layers. Cephalothin 1 g was administered intramuscularly twice daily for 3 days postoperatively. The animals were kept alive for 150 days without any other form of treatment.
The mean transvalvular gradient, effective orifice area, and the mobility and competence of the valvular leaflets were determined by two-dimensional echocardiography. The hemodynamic evaluation was completed with angiography at the time of sacrifice. The animals were anesthetized and right and left catheterization was performed through the right jugular vein and right carotid artery to determine the mean systemic and pulmonary pressure, pulmonary artery wedge pressure, and cardiac output. Radiologic evaluation was undertaken using mammography. The explanted valves were evaluated for the presence of macroscopic foci of calcification. For histologic and immunohistochemical evaluation, the retrieved valves were fixed in HistoChoice (Amresco, Inc., Solon, OH, USA) and sectioned into fragments including the base, midportion, and free edge of the leaflets. The fragments were embedded in paraffin, stained in hematoxylin-eosin and von Kossa, and examined with an Olympus CBA microscope (Olympus, Tokyo, Japan). The valvular ultrastructure was examined by transmission electron microscopy with an EM 301 electron microscope (Philips, Drachten, The Netherlands). The remnants of the leaflets were dehydrated with acetone to the critical point of carbon dioxide, coated with gold, and examined in a scanning electron microscope (Stereoscam 240; Cambridge Instruments, Cambridge, UK) for endothelial repopulation. The viability of the endothelial cells was determined by immunohistochemical staining with factor VIII (polyclonal von Willebrand factor; Dako, Grostrup, Denmark).
Quantitative variables were expressed as mean ± standard error of the mean. Comparisons between groups were performed with the unpaired Student t test. Qualitative variables were described as absolute and relative frequencies, and compared by Fisher’s exact test. Statistical significance was established at p < 0.05. The assumptions of a normal distribution and equal variances were tested. Data and statistical analyses were performed with Graphpad InStat 3.0 for Windows 95 (GraphPad Software, San Diego, CA, USA).
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RESULTS
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On echocardiographic and hemodynamic evaluation, no regurgitation was seen in either group, but in one control, decreased leaflet mobility was noted due to severe calcification. No statistical differences existed between groups regarding transvalvular gradients, effective orifice area, systemic and pulmonary pressures, and cardiac output (Table 1
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Table 1. Echocardiographic and Hemodynamic Variables in Sheep with L-Hydro-treated (Test) and Glutaraldehyde-treated (Control) Mitral Valve Bioprostheses
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Macroscopically, there was no evidence of endocarditis, thrombosis, hematomas, abrasions, or structural lesions in the test group. An organized thrombus and an extensive area of calcification were seen on the inflow surfaces of all 3 GA-fixed valves. Calcium deposits were also detected by mammography in all GA-fixed valves; these were not observed in the L-Hydro-treated valves. Optical microscopy revealed nearly complete coverage by endothelial cells of the surfaces of all the L-Hydro-treated valves, except on the tips of the leaflets. This new endothelium was in direct contact with the underlying collagen layer (Figure 1). In contrast, fibrin deposits, macrophages, calcium, and thrombotic material covered the surfaces of the GA-treated valves; only sparse endothelial cells were observed, and their contact with the underlying tissue was incomplete. The von Kossa stain confirmed the presence of calcification in these valves.
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Figure 1. Hematoxylin-eosin stain showing the surface of an L-Hydro-treated valve covered by endothelial cells, with fibroblasts incorporated into the valvular matrix.
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Scanning electron microscopy demonstrated a confluent layer of cells with the typical morphology (cobblestone) of endothelial cells, which covered almost the entire surface of the L-Hydro-treated valves (Figure 2). These cells were seen only at the base of the GA-fixed leaflets, which showed extensive areas of denuded collagen (Figure 3). Transmission electron microscopy indicated viable fibroblasts repopulating the collagen matrix of the L-Hydro valves, which were not found in the control group. The expression of factor VIII confirmed by hematoxylin-eosin staining in the L-Hydro-treated valves demonstrated that the cellular coverage was composed of viable endothelial cells (Figure 4). Weak factor VIII expression was present in the GA-fixed valves. The histologic findings after 150 days of valvular implantation are summarized in Table 2.
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Figure 2. Scanning electron microscopy showing a layer of confluent endothelial cells in L-Hydro-treated valves (original magnification x 1,500).
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Figure 3. Scanning electron microscopy showing a lack of endothelial cells and denuded collagen in glutaraldehyde-fixed leaflets (original magnification x 2,500).
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DISCUSSION
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The main disadvantage of heart valve bioprostheses is their limited long-term durability due to tissue degeneration, which has been identified as the result of an immunogenic reaction to the xenogeneic tissue caused by the cytotoxicity of glutaraldehyde.9 Glutaraldehyde reduces the relaxation and increases the stiffness of the leaflets, leading to abnormal mechanical function of the valvular prostheses, which results in localized tissue damage that ultimately promotes thrombogenicity.10–12 Currently, it is believed that development of a protective layer of endothelial cells can assure greater resistance and durability of valve bioprostheses.13–14 However, growth of endothelium on the surface of the leaflets occurs only if they are processed by an atoxic method of tissue preservation. Most commercial bioprostheses are preserved with glutaraldehyde that prevents endothelial repopulation.9 Ishihara and colleagues15 demonstrated that only 23% of biological valves explanted during a 12 to 60 month period had evidence of endothelial coverage, and this was restricted to the base of the leaflets. This incapacity for re-endothelialization makes the leaflets vulnerable to the leaching of proteins and salt, which ultimately leads to calcification.16
In this study, the non-GA-preserved valves proved more resistant to thrombosis, calcification, inflammation, and mechanical wear. Calcification was more evident in the GA-fixed valves, causing a reduction in leaflet mobility and consequently increased (although not significant) mean pulmonary artery pressure and pulmonary artery wedge pressure. This is in accordance with the finding in a study by Schoen and colleagues17 who used the same experimental model (juvenile sheep) and a similar length of observation (150 days).
Re-endothelialization was not observed in the control group, except for a pannus on the base of the leaflets, with incomplete contact with the underlying collagen matrix. The viability of the endothelial cells could not be clearly demonstrated by factor VIII in this group. On the other hand, the L-Hydro-treated valves demonstrated spontaneous endothelialization with evidence of strong cell attachment and viability, as described for valves treated by enzymatic decellularization and photooxidation.18–19
Previous studies have demonstrated that recellularization of the surface and collagen matrix with host endothelial cells promotes regeneration and growth of the valvular tissue creating a living structure.4,16 Some alternatives, such as in vitro endothelial seeding with recipient cells, have been tried; however, this method still has some limitations, for example, the need for intervention to obtain endothelial cells, cellular growth in a culture medium, weak endothelium-collagen attachment, and low resistance to shear stress of the new endothelium.13,20 The technique used in this study, of controlled extraction of antigenic substances, makes the matrix cyto-compatible, keeping the physical properties of the leaflets unchanged. The spontaneous recellularization is similar to natural tissue regeneration and avoids the pitfalls of in vitro re-endothelialization. The results indicate that L-Hydro-treated porcine heart valve tissues are capable of spontaneous endothelialization with strong cell attachment of the new endothelium to the collagen tissue matrix. This capacity for re-endothelialization with host cells is a promising means of improving function and long-term durability of bioprosthetic valves.
Presented at the 2nd Biennial Meeting of the Society for Heart Valve Disease. Paris, France, June 28 – July 1, 2003.
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ACKNOWLEDGMENTS
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The authors thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for its financial support in this research (Process n° 2002/08298-4).
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REFERENCES
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ABSTRACT
INTRODUCTION
