Tissue Engineering of Heart Valves
http://www.100md.com
《循环学杂志》
the Department of Cardiothoracic Surgery and Ludwig-Boltzmann-Institute for Cardiosurgical Research, Medical University of Vienna, Vienna, Austria.
Abstract
Background— Tissue-engineered or decellularized heart valves have already been implanted in humans or are currently approaching the clinical setting. The aim of this study was to examine the migratory response of human monocytic cells toward decellularized porcine and human heart valves, a pivotal step in the early immunologic reaction.
Methods and Results— Porcine and human pulmonary valve conduits were decellularized, and migration of U-937 monocytic cells toward extracted heart valve proteins was examined in a transmigration chamber in vitro. Homogenized tissue specimens were size fractionated by SDS-PAGE. The decellularization procedure effectively reduced the migration of human monocytes toward all heart valve tissue. However, only the antigen reduction of human pulmonary valves abolished the monocytic response (wall, 0.88±0.19% versus 30.20±3.93% migrated cells [mean±SEM]; cusps, 0.10±0.06% versus 10.24±1.83%) and was significantly lower (P<0.05) than that of the decellularized porcine equivalent (wall, 5.03±0.14% versus 24.31±2.38%; cusps, 3.18±0.38% versus 10.24±1.83%). SDS-PAGE of the pulmonary heart valve tissue revealed that considerable amounts of proteins with different molecular weights that were not detected in the human equivalent remain in the decellularized porcine heart valve.
Conclusions— We describe for the first time that the remaining potential of decellularized pulmonary heart valves to attract monocytic cells depends strongly on whether porcine or human scaffolds were used. These findings will have an important impact on further investigations in the field of heart valve tissue engineering.
Key Words: immunology ; inflammation ; tissue engineering ; valves
Introduction
Xenograft heart valves of porcine origin treated with glutaraldehyde and mounted in a rigid stent are the most widely used cardiac bioprostheses. Although stable cross-links in cellular and extracellular matrix proteins are considered to reduce immunogenicity, glutaraldehyde treatment has been demonstrated to increase calcification by fixing cellular debris in place.1,2 In addition, the residual ability of the porcine tissue to trigger an immune response may activate macrophages, which can obtain an osteoblast calcium-depositing phenotype.3 Long-term durability of these nonviable valves provided unsatisfactory results. Tissue-engineered heart valves constructed from a xenogeneic or an allogeneic heart valve scaffold eventually covered with autologous endothelial cells are considered to overcome these obstacles and have already been implanted in humans.4,5
See p 2712
We previously reported the effective decellularization of porcine heart valve tissue.6 This study presents a further important evaluation focusing on the monocytic response against decellularized heart valves, which, to the best of our knowledge, has not been investigated to date.
It is well described that peripheral blood monocytes interact with the endothelial lining as an initial step in immune reactions. So that the movement of monocytes from blood into tissue occurs in a site-directed manner, focal factors determine the capture of circulating leukocytes. Once arrested at the endothelial surface, leukocytes may be directed by a chemoattractant gradient to transmigrate into the perivascular tissue. Chemotactic factor–induced migration of monocytes was shown to be independent of other cell types.
To investigate the possibility that decellularization procedures for porcine heart valves alter the migratory response of monocytic cells toward the xenogenic tissue, we have used an in vitro migration assay. Differentiated U-937 cells, a human monoblast cell line, have been used as a model to study the migratory function of human monocytes. Using this approach, we can demonstrate that even in decellularized tissue, a residual soluble gradient of extractable porcine heart valve proteins leads to a rapid migration of U-937 cells across a polyethylene-terephtalate (PET) membrane bearing 3-μm pores. Furthermore, we describe for the first time the finding that decellularized porcine and human pulmonary heart valves differ importantly in their remaining potential to attract monocytic cells.
Methods
Decellularization Procedure
Porcine pulmonary heart valve conduits (n=12) of pigs (80 to 100 kg) obtained from a local slaughterhouse were excised of adherent fat and myocardium, leaving only a small subvalvular muscle bulge. The valve diameter was 18 to 20 mm. Within 2 hours after explantation, conduits were incubated in sterile water containing vancomycin (12 mg/L), metronidazol (12 mg/L), amikacin (12 mg/L), and ciprofloxacin (3 mg/L) for 48 hours at 4°C. Six porcine pulmonary heart valves were treated with a modified detergent-based decellularization procedure.6 Briefly, the conduits were placed into 100 mL sterile water containing 0.05% tert-octylphenyl-polyoxyethylen (Triton X-100, Biorad), 0.05% sodium-deoxycholate (Merck), and 0.05% octylphenyl-polyethylene glycol (IGEPAL-CA630, ICN) for 48 hours at 4°C. Conduits were subsequently treated with ribonuclease (RNase, 100 μg/mL, Roche Diagnostics GmbH) and deoxyribonuclease (DNase, 150 IU/mL, Sigma) with 50 mmol/L MgCl2 in PBS (Ca2+ and Mg2+ free [PBS–/–], Gibco) for 24 hours at 37°C. To remove residual detergents, the heart valve conduits were subsequently washed for 12 days in PBS–/– that was changed every second day, with an additional antibiotic sterilization carried out for 3 days at the end of the washing procedure. All steps were conducted under continuous shaking.
Human pulmonary heart valves were obtained in the course of heart transplantations. Valve conduits that could not be used for implantation as homograft substitutes for several reasons were either cryopreserved after antibiotic sterilization (n=4) or decellularized (n=4) with the procedure described above.
Two longitudinal slices (5 mm) of each conduit (including leaflet) were excised for histology (n=40). Tissue specimens of the remaining native and decellularized porcine and cryopreserved and decellularized human pulmonary wall and leaflet tissue were snap-frozen in liquid nitrogen and stored at –80°C until use for protein extraction and electrophoresis.
Histology
For detection of tissue integrity and cell removal, histological examination was performed. The 2 longitudinal slices excised from each conduit were embedded in paraffin. For general morphology, 3 sections (10 μm) of each slice were stained with hematoxylin-eosin (HE) and examined by 2 independent assistants using high-power field light microscopy. Immunohistostaining was carried out with immunofluorescence techniques. Primary antibodies were polyclonal anti-porcine collagen type I and III (polyclonal rabbit IgG, 1:20, Monosan) cross-reactive with human type I and III collagen and monoclonal anti-elastin (1:2000, monoclonal anti-elastin from mouse ascites, Clone BA-4, Sigma). The DNA-specific dye TOPRO-3 (Molecular Probes) was used to detect remaining cell nuclei in the heart valve scaffold.
Three sections of each slice were triple stained and analyzed by confocal laser scanning microscopy (ZEISS LSM 510 laser scanning microscope, Jena) using a 3-dimensional analysis mode.
Preparation of Tissue Extracts
The frozen tissue samples were weighed and washed with cold PBS–/–. Exactly 100 mg of porcine or human pulmonary wall and leaflet tissue was homogenized in 2 mL ice-cold DMEM (Bio Whittaker) using a mortar and pestle. The homogenization was carried out mechanically at 700 rpm for 10 minutes on ice. Sterile technique was used throughout the experiments. The homogenates were aspirated and transferred into centrifuge tubes, and the debris of each homogenate was sedimented by subsequent centrifugation at 10 000g and 18°C for 30 minutes. Then, 1.6 mL of each supernatant was carefully withdrawn. Next, 100 μL of each sample was drawn, and the protein content was determined by a modified Bradford assay (Biorad). The remaining 1.5 mL was used for migration experiments. DMEM without protein extracts (1.5 mL) was used as negative control.
U-937 Mononuclear Phagocytes Cell Culture
U-937 monocytic cells7 were purchased from ATCC and cultivated in RPMI 1640 medium supplemented with 10% FCS (Promocell) and antibiotics at 37°C and 5% CO2. Fresh medium was added every 4 days, and cell number was determined with a Microdiff hematology analyzer (Coulter). For differentiation to the monocyte/macrophage phenotype, the cells were treated with 10 nmol/L 1.25-(OH)2-Calcitriol8 for 72 hours. Before plating into assay chambers, cells were washed 2 times with DMEM to remove FCS and antibiotics and subsequently resuspended in DMEM without any supplements to a final concentration of 1.0x106 cells/mL.
Monocyte Migration Assay
The in vitro cell migration assays were performed with a 6-well culture plate with a PET membrane (Falcon cell culture inserts, Beckton Dickinson Labware) placed in each well. To minimize random migration of U-937 cells, the pore size of the membrane used in all experimental settings was 3 μm. The cell inserts divided the wells into a lower (LCH) and an upper (UCH) chamber. The bottom of the wells was covered with 1.5 mL of the protein extracts of heart valve specimens or negative control medium. We placed 1.5x106 U-937 cells onto the upper side of the filter. The chambers were incubated in a humidified 5% CO2 atmosphere for 24 hours at 37°C. After the incubation period, the cell inserts were removed, and the bottom adhesive cells were loosened with a cell scraper. The transmigrated U-937 cells were centrifuged at 300g for 10 minutes, and the supernatant was discarded. Cells were stained by adding 200 μL crystal violet solution and counted by an independent assistant using a hemocytometer.
To examine whether an altered migratory response is caused by the decellularization procedure or simply depends on a reduced protein content of the extracts added into the LCH, protein extracts of smaller (40 mg) native porcine and homograft pulmonary wall specimens were prepared, again using 2 mL ice-cold DMEM for homogenization. Tissue extracts were prepared as described above, and the migratory response was compared with that of conventionally used 100-mg specimens.
Protein Electrophoresis
Approximately 200 mg of the native and decellularized heart valves was grinded in 1 mL ice-cold DMEM on ice, and the total protein content of the homogenized tissue was determined as described above. Aliquots of grinded tissue (75 μg protein) were mixed with equal volumes of SDS-PAGE sample buffer on ice and then boiled for 3 minutes. Samples were subjected to electrophoresis on a 10% (wt/vol) SDS-PAGE gel overlaid with a 4% (wt/vol) acrylamide stacking gel. After staining with Coomassie Brilliant Blue R-250, the gels were scanned for electronic image editing. To detect even weakly visible protein bands, brightness and contrast were optimized with Adobe Photoshop 5.0 Software.
Statistical Analysis
All values are shown as mean±SEM. The Wilcoxon rank-sum test was used to assess differences between groups. Values of P<0.05 were considered significant.
Results
Decellularization Procedure
After the decellularization process, no cells could be detected by HE staining within the examined porcine or human valve tissue sections (n=120; representative photomicrographs of porcine conduit wall and leaflets are shown in Figure 1A and 1B). The DNA-specific TO-PRO 3 staining also revealed a heart valve scaffold free of cell nuclei, indicating effective removal of cells within the porcine pulmonary conduit wall and cusps. As shown by representative laser scanning micrographs, collagen and elastin composition was preserved within the heart valve after the decellularization procedure (representative photomicrographs of porcine conduit wall and leaflets are shown in Figure 1C and 1D).
Migration Assay
Compared with the negative control (10.3±2.1x103 cells in LCH; n=12), a strongly increased migration of U-937 cells across the PET membrane was seen within 24 hours when protein extracts of native porcine pulmonary wall (364.5± 59.6x103; n=10; P<0.01) or the homograft conduit wall (452.9±58.9x103; n=10; P<0.01) were added into the LCH of the transmigration chamber (Figure 2A). These data indicate that a gradient of extracted soluble valve tissue proteins leads to the migration of monocytic cells.
Compared with conventional homograft arterial wall tissue, the proteins of the decellularized porcine pulmonary artery caused a significantly decreased (75.4±2.1x103; n=10; P<0.01) migration of monocytic cells. However, the migration toward decellularized human pulmonary arterial wall tissue (13.1±2.8x103; n=10) was significantly lower (P<0.01) than that of the porcine equivalent and comparable to the negative control (Figure 2A).
Even more surprising results were seen when the migratory response toward the extracts of the heart valve cusps was examined (Figure 2B). Whereas the proteins extracted from native porcine leaflet specimens initiated 153.7± 27.7x103 cells to transmigrate across the membrane, the decellularization procedure reduced the number of U-937 cells in the LCH to 47.7±5.7x103. The extracts of the human native pulmonary cusps, however, already elicited a smaller amount of 54.4±11.4x103 monocytic cells to transmigrate toward the heart valve proteins. The decellularized human pulmonary valve leaflets again led only 2.1±0.9x103 cells to migrate through the membrane, which was within the range of random migration (range, 1 to 19x103 cells in LCH).
Although the smaller specimens (40 mg; n=7) of the native porcine pulmonary wall revealed a proportionally lower concentration of extracted proteins (40 mg, 0.35±0.04 μg/μL versus 100 mg, 0.83±0.10 μg/μL; P<0.01), the migratory response was similar (40 mg, 22.27±2.57% transmigrated cells versus 100 mg, 24.31±2.38%); 0.36±0.03 μg/μL proteins were extracted from the decellularized porcine conduit wall, but only 5.03±0.14% of U-937 cells were attracted to transmigrate toward the extracts. Similar results were seen when the differently sized specimens of the homograft pulmonary wall were compared (Table). These data indicate that the reduced migration of monocytes truly is caused by the decellularization procedure and does not simply result from the lower concentration of extracted proteins in the LCH.
Weight and Extracted Proteins of Pulmonary Conduit Wall and Cusps and Percentage of Transmigrated Monocytic Cells
Protein Electrophoresis
Electrophoretic separation of homogenized pulmonary cusp and conduit wall tissue demonstrated that the decellularization procedure removes considerable amounts of proteins within the porcine and human heart valves, indicating the effective reduction of potential immunogenic components (Figure 3A and 3B). However, within the examined molecular weight range of 25 to 250 kDa, protein bands were still detectable by SDS-PAGE within the decellularized porcine pulmonary wall tissue. Even the much thinner porcine leaflet tissue revealed residual proteins after the decellularization procedure. In contrast, fewer protein bands were detected in the homogenates of the decellularized human pulmonary wall and leaflet tissue.
Discussion
The durability of conventional glutaraldehyde-treated biological heart valve substitutes is limited because of early calcification or degeneration.9 It is well known that cells or cell membranes within the tissue provoke early calcification of biological heart valve prostheses10,11 whereby a direct connection between specific antibody response and porcine valve tissue calcification has been demonstrated.12 That homograft heart valves elicit a host-dependent immune response against the implanted tissue has been well described.13,14 The concept of decellularization or tissue engineering of heart valves was developed and modified by several groups to circumvent these problems.15–18 Tissue engineering is defined as the regeneration of biological tissue through the use of cells with the aid of supporting structures and/or biomolecules.
Decellularized allogeneic valve prostheses eventually covered with endothelial cells have already been used clinically in humans.4,5 Immunologic evaluations demonstrated strongly decreased antibody response in vivo and satisfactory results in the short term.19–21 Although a decellularization of xenogenous heart valves is also considered to diminish antigenic content,4 early failure and strong inflammatory response against implants were described. The implantation of decellularized porcine heart valves in humans resulted in fibrous sheath formation outside and inside the graft. The histological examination demonstrated a dense neutrophil granulocyte and macrophage reaction around the graft with infiltration of the leaflet tissue,22 which likely represents a standard foreign body–type reaction in which the body fails to remove and consequently tries to encapsulate foreign material. These data raise the suspicion that the decellularized porcine heart valve is not as weakly immunogenic as it is considered to be, and it is highly likely that seeding with endothelial cells might not prevent the immigration of immune competent cells in vivo.
Focusing on the elimination of immunogenic components within a porcine heart valve showed that the detergent-based decellularization procedure revealed no histologically detectable residual cells or cell nuclei. To date, it is unclear to what extent a decellularization procedure needs to remove noncellular integral components of the extracellular matrix like glycosaminoglycans. Because of their ability to interact with cytokines and chemokines, their removal might play an important role in preventing any immune response.23 On the other hand, glycosaminoglycans also appear to play a significant role in tissue elasticity and the prevention of calcification.24 These controversies need to be examined in further studies. Recently, it has been demonstrated in a sheep model that the inflammatory reaction against decellularized heart valves plays a crucial role in valve deterioration.25 The initial events of inflammatory response involve a variety of cell types, including monocytes, and degradation products of extracellular matrix proteins. Monocyte migration is a highly regulated process in which these motile cells sense and respond directionally to chemical gradients, which results in immigration of mononuclear cells at sites of immune response.26
Although U-937 cells represent a transformed monoblastoid cell line, their in vitro behavior parallels that of differentiating mononuclear phagocytes, and their response to chemotactic stimuli is well characterized.27 Using this monocytic cell line for an in vitro migration assay, we could examine the response of human monocytic cells toward decellularized pulmonary heart valve tissue under standardized conditions. We could clearly show that the decellularization of porcine or human heart valves significantly reduces their potential to attract monocytic cells. It is shown that even a much lower protein concentration of native valve specimens still provoked a much higher cell migration than did the extracts of the decellularized heart valve. Thus, it could be proved that the reduced migratory response toward the valve tissue is truly caused by the antigen reduction procedure. It has already been presented that a decellularization process strongly decreases the humoral response in vivo; this study presents a further step in the immunologic evaluation of heart valve substitutes.
However, the porcine pulmonary heart valve treated by the decellularization procedure did not reveal the considered nonantigenic heart valve scaffold. Unexpectedly, only the extracts of the decellularized human pulmonary heart valve did not provoke any monocytic migratory response in vitro. Furthermore, the extracts of the decellularized porcine pulmonary cusps elicited a comparable monocytic migratory response as the untreated cellular homograft leaflets, known to initiate an early host response of inflammatory cells in vivo, predominantly consisting of macrophages.28 Again, the decellularization of human pulmonary heart valves abolished the chemotactic activity of the leaflet tissue.
SDS-PAGE revealed that the antigen reduction procedure effectively removes considerable amounts of proteins within the decellularized heart valve. However, even after the histologically verified effective decellularization procedure, considerable amounts of proteins were still detectable within the xenogenous conduit wall that were not detected within the human equivalent. These remaining proteins may provoke the migratory response seen. Even more surprisingly, we observed that the decellularized porcine heart valve cusps initiated an amount of monocytic cells to transmigrate across the membrane similar to that of the native human cusp, although fewer residual xenogenous proteins were detected by electrophoretic seperation. These data demonstrate that the porcine heart valve treated by an decellularization procedure is not as weakly immunogenic as it is considered to be. The detailed composition of the residual xenogeneic components within the porcine extracellular matrix and the proteins leading to monocyte migration has to be elucidated in further studies currently being carried out in our laboratories.
Because heart valve prostheses have to withstand strong biomechanical stress in vivo from the point of implantation, an enhanced early inflammatory reaction could lead to a significant weakening of the heart valve scaffold and might be the cause of graft rupture.22
In conclusion, we demonstrate for the first time that the migratory response of monocytic cells toward heart valves treated by a decellularization process is significantly different in scaffolds of porcine and human origin. Whereas the decellularized porcine pulmonary valve does not represent a completely nonimmunogenic heart valve scaffold, the decellularization of a human pulmonary heart valve strongly diminishes the migration of monocytic cells toward the valve tissue. Because some of these new biological valve prostheses have already been implanted in humans and others are currently approaching the clinical setting, these findings will have an important impact on further investigations of tissue-engineered heart valves.
References
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Rieder E, Kasimir MT, Silberhumer G, Seebacher G, Wolner E, Simon P, Weigel G. Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J Thorac Cardiovasc Surg. 2004; 172: 399–405.
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O’Brien MF, Goldstein S, Walsh S, Black KS, Elkins R, Clarke D. The Synergraft: a new acellular (non-GA-fixed) tissue heart valve for autologous recellularization: first experimental studies before clinical implantation. Semin Thorac Cardiovasc Surg. 1999; 11 (suppl 1): 194–200.
Stock UA, Nagashima M, Khalil PN, Nollert GD, Herden T, Sperling JS, Moran A, Lien J, Martin DP, Schoen FJ, Vacanti JP, Mayer JE Jr. Tissue-engineered valve conduits in the pulmonary circulation. J Thorac Cardiovasc Surg. 2000; 119 (pt 1): 732–740.
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Hawkins JA, Hilman ND, Lambert L, Jones J, Di Russo GB, Profaizer T, Fuller TC, Minich LL, Williams RV, Shaddy RE. Immunogenicity of decellularized cryopreserved allografts in pediatric cardiac surgery: comparison with standard cryopreserved allografts. J Thorac Cardiovasc Surg. 2003; 126: 247–253.
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Simon P, Kasimir MT, Seebacher G, Weigel G, Ullrich R, Salzer-Muhar U, Rieder E, Wolner E. Early failure of the tissue engineered porcine heart valve SynergraftTM in pediatric patients. Eur J Cardiothorac Surg. 2003; 23: 1002–1006.
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Koolbergen DR, Hazekamp MG, de Heer E. The pathology of fresh and cryopreserved homograft heart valves: an analysis of forty explanted homograft valves. J Thorac Cardiovasc Surg. 2002; 124: 689–697.(Erwin Rieder, MD; Gernot )
Abstract
Background— Tissue-engineered or decellularized heart valves have already been implanted in humans or are currently approaching the clinical setting. The aim of this study was to examine the migratory response of human monocytic cells toward decellularized porcine and human heart valves, a pivotal step in the early immunologic reaction.
Methods and Results— Porcine and human pulmonary valve conduits were decellularized, and migration of U-937 monocytic cells toward extracted heart valve proteins was examined in a transmigration chamber in vitro. Homogenized tissue specimens were size fractionated by SDS-PAGE. The decellularization procedure effectively reduced the migration of human monocytes toward all heart valve tissue. However, only the antigen reduction of human pulmonary valves abolished the monocytic response (wall, 0.88±0.19% versus 30.20±3.93% migrated cells [mean±SEM]; cusps, 0.10±0.06% versus 10.24±1.83%) and was significantly lower (P<0.05) than that of the decellularized porcine equivalent (wall, 5.03±0.14% versus 24.31±2.38%; cusps, 3.18±0.38% versus 10.24±1.83%). SDS-PAGE of the pulmonary heart valve tissue revealed that considerable amounts of proteins with different molecular weights that were not detected in the human equivalent remain in the decellularized porcine heart valve.
Conclusions— We describe for the first time that the remaining potential of decellularized pulmonary heart valves to attract monocytic cells depends strongly on whether porcine or human scaffolds were used. These findings will have an important impact on further investigations in the field of heart valve tissue engineering.
Key Words: immunology ; inflammation ; tissue engineering ; valves
Introduction
Xenograft heart valves of porcine origin treated with glutaraldehyde and mounted in a rigid stent are the most widely used cardiac bioprostheses. Although stable cross-links in cellular and extracellular matrix proteins are considered to reduce immunogenicity, glutaraldehyde treatment has been demonstrated to increase calcification by fixing cellular debris in place.1,2 In addition, the residual ability of the porcine tissue to trigger an immune response may activate macrophages, which can obtain an osteoblast calcium-depositing phenotype.3 Long-term durability of these nonviable valves provided unsatisfactory results. Tissue-engineered heart valves constructed from a xenogeneic or an allogeneic heart valve scaffold eventually covered with autologous endothelial cells are considered to overcome these obstacles and have already been implanted in humans.4,5
See p 2712
We previously reported the effective decellularization of porcine heart valve tissue.6 This study presents a further important evaluation focusing on the monocytic response against decellularized heart valves, which, to the best of our knowledge, has not been investigated to date.
It is well described that peripheral blood monocytes interact with the endothelial lining as an initial step in immune reactions. So that the movement of monocytes from blood into tissue occurs in a site-directed manner, focal factors determine the capture of circulating leukocytes. Once arrested at the endothelial surface, leukocytes may be directed by a chemoattractant gradient to transmigrate into the perivascular tissue. Chemotactic factor–induced migration of monocytes was shown to be independent of other cell types.
To investigate the possibility that decellularization procedures for porcine heart valves alter the migratory response of monocytic cells toward the xenogenic tissue, we have used an in vitro migration assay. Differentiated U-937 cells, a human monoblast cell line, have been used as a model to study the migratory function of human monocytes. Using this approach, we can demonstrate that even in decellularized tissue, a residual soluble gradient of extractable porcine heart valve proteins leads to a rapid migration of U-937 cells across a polyethylene-terephtalate (PET) membrane bearing 3-μm pores. Furthermore, we describe for the first time the finding that decellularized porcine and human pulmonary heart valves differ importantly in their remaining potential to attract monocytic cells.
Methods
Decellularization Procedure
Porcine pulmonary heart valve conduits (n=12) of pigs (80 to 100 kg) obtained from a local slaughterhouse were excised of adherent fat and myocardium, leaving only a small subvalvular muscle bulge. The valve diameter was 18 to 20 mm. Within 2 hours after explantation, conduits were incubated in sterile water containing vancomycin (12 mg/L), metronidazol (12 mg/L), amikacin (12 mg/L), and ciprofloxacin (3 mg/L) for 48 hours at 4°C. Six porcine pulmonary heart valves were treated with a modified detergent-based decellularization procedure.6 Briefly, the conduits were placed into 100 mL sterile water containing 0.05% tert-octylphenyl-polyoxyethylen (Triton X-100, Biorad), 0.05% sodium-deoxycholate (Merck), and 0.05% octylphenyl-polyethylene glycol (IGEPAL-CA630, ICN) for 48 hours at 4°C. Conduits were subsequently treated with ribonuclease (RNase, 100 μg/mL, Roche Diagnostics GmbH) and deoxyribonuclease (DNase, 150 IU/mL, Sigma) with 50 mmol/L MgCl2 in PBS (Ca2+ and Mg2+ free [PBS–/–], Gibco) for 24 hours at 37°C. To remove residual detergents, the heart valve conduits were subsequently washed for 12 days in PBS–/– that was changed every second day, with an additional antibiotic sterilization carried out for 3 days at the end of the washing procedure. All steps were conducted under continuous shaking.
Human pulmonary heart valves were obtained in the course of heart transplantations. Valve conduits that could not be used for implantation as homograft substitutes for several reasons were either cryopreserved after antibiotic sterilization (n=4) or decellularized (n=4) with the procedure described above.
Two longitudinal slices (5 mm) of each conduit (including leaflet) were excised for histology (n=40). Tissue specimens of the remaining native and decellularized porcine and cryopreserved and decellularized human pulmonary wall and leaflet tissue were snap-frozen in liquid nitrogen and stored at –80°C until use for protein extraction and electrophoresis.
Histology
For detection of tissue integrity and cell removal, histological examination was performed. The 2 longitudinal slices excised from each conduit were embedded in paraffin. For general morphology, 3 sections (10 μm) of each slice were stained with hematoxylin-eosin (HE) and examined by 2 independent assistants using high-power field light microscopy. Immunohistostaining was carried out with immunofluorescence techniques. Primary antibodies were polyclonal anti-porcine collagen type I and III (polyclonal rabbit IgG, 1:20, Monosan) cross-reactive with human type I and III collagen and monoclonal anti-elastin (1:2000, monoclonal anti-elastin from mouse ascites, Clone BA-4, Sigma). The DNA-specific dye TOPRO-3 (Molecular Probes) was used to detect remaining cell nuclei in the heart valve scaffold.
Three sections of each slice were triple stained and analyzed by confocal laser scanning microscopy (ZEISS LSM 510 laser scanning microscope, Jena) using a 3-dimensional analysis mode.
Preparation of Tissue Extracts
The frozen tissue samples were weighed and washed with cold PBS–/–. Exactly 100 mg of porcine or human pulmonary wall and leaflet tissue was homogenized in 2 mL ice-cold DMEM (Bio Whittaker) using a mortar and pestle. The homogenization was carried out mechanically at 700 rpm for 10 minutes on ice. Sterile technique was used throughout the experiments. The homogenates were aspirated and transferred into centrifuge tubes, and the debris of each homogenate was sedimented by subsequent centrifugation at 10 000g and 18°C for 30 minutes. Then, 1.6 mL of each supernatant was carefully withdrawn. Next, 100 μL of each sample was drawn, and the protein content was determined by a modified Bradford assay (Biorad). The remaining 1.5 mL was used for migration experiments. DMEM without protein extracts (1.5 mL) was used as negative control.
U-937 Mononuclear Phagocytes Cell Culture
U-937 monocytic cells7 were purchased from ATCC and cultivated in RPMI 1640 medium supplemented with 10% FCS (Promocell) and antibiotics at 37°C and 5% CO2. Fresh medium was added every 4 days, and cell number was determined with a Microdiff hematology analyzer (Coulter). For differentiation to the monocyte/macrophage phenotype, the cells were treated with 10 nmol/L 1.25-(OH)2-Calcitriol8 for 72 hours. Before plating into assay chambers, cells were washed 2 times with DMEM to remove FCS and antibiotics and subsequently resuspended in DMEM without any supplements to a final concentration of 1.0x106 cells/mL.
Monocyte Migration Assay
The in vitro cell migration assays were performed with a 6-well culture plate with a PET membrane (Falcon cell culture inserts, Beckton Dickinson Labware) placed in each well. To minimize random migration of U-937 cells, the pore size of the membrane used in all experimental settings was 3 μm. The cell inserts divided the wells into a lower (LCH) and an upper (UCH) chamber. The bottom of the wells was covered with 1.5 mL of the protein extracts of heart valve specimens or negative control medium. We placed 1.5x106 U-937 cells onto the upper side of the filter. The chambers were incubated in a humidified 5% CO2 atmosphere for 24 hours at 37°C. After the incubation period, the cell inserts were removed, and the bottom adhesive cells were loosened with a cell scraper. The transmigrated U-937 cells were centrifuged at 300g for 10 minutes, and the supernatant was discarded. Cells were stained by adding 200 μL crystal violet solution and counted by an independent assistant using a hemocytometer.
To examine whether an altered migratory response is caused by the decellularization procedure or simply depends on a reduced protein content of the extracts added into the LCH, protein extracts of smaller (40 mg) native porcine and homograft pulmonary wall specimens were prepared, again using 2 mL ice-cold DMEM for homogenization. Tissue extracts were prepared as described above, and the migratory response was compared with that of conventionally used 100-mg specimens.
Protein Electrophoresis
Approximately 200 mg of the native and decellularized heart valves was grinded in 1 mL ice-cold DMEM on ice, and the total protein content of the homogenized tissue was determined as described above. Aliquots of grinded tissue (75 μg protein) were mixed with equal volumes of SDS-PAGE sample buffer on ice and then boiled for 3 minutes. Samples were subjected to electrophoresis on a 10% (wt/vol) SDS-PAGE gel overlaid with a 4% (wt/vol) acrylamide stacking gel. After staining with Coomassie Brilliant Blue R-250, the gels were scanned for electronic image editing. To detect even weakly visible protein bands, brightness and contrast were optimized with Adobe Photoshop 5.0 Software.
Statistical Analysis
All values are shown as mean±SEM. The Wilcoxon rank-sum test was used to assess differences between groups. Values of P<0.05 were considered significant.
Results
Decellularization Procedure
After the decellularization process, no cells could be detected by HE staining within the examined porcine or human valve tissue sections (n=120; representative photomicrographs of porcine conduit wall and leaflets are shown in Figure 1A and 1B). The DNA-specific TO-PRO 3 staining also revealed a heart valve scaffold free of cell nuclei, indicating effective removal of cells within the porcine pulmonary conduit wall and cusps. As shown by representative laser scanning micrographs, collagen and elastin composition was preserved within the heart valve after the decellularization procedure (representative photomicrographs of porcine conduit wall and leaflets are shown in Figure 1C and 1D).
Migration Assay
Compared with the negative control (10.3±2.1x103 cells in LCH; n=12), a strongly increased migration of U-937 cells across the PET membrane was seen within 24 hours when protein extracts of native porcine pulmonary wall (364.5± 59.6x103; n=10; P<0.01) or the homograft conduit wall (452.9±58.9x103; n=10; P<0.01) were added into the LCH of the transmigration chamber (Figure 2A). These data indicate that a gradient of extracted soluble valve tissue proteins leads to the migration of monocytic cells.
Compared with conventional homograft arterial wall tissue, the proteins of the decellularized porcine pulmonary artery caused a significantly decreased (75.4±2.1x103; n=10; P<0.01) migration of monocytic cells. However, the migration toward decellularized human pulmonary arterial wall tissue (13.1±2.8x103; n=10) was significantly lower (P<0.01) than that of the porcine equivalent and comparable to the negative control (Figure 2A).
Even more surprising results were seen when the migratory response toward the extracts of the heart valve cusps was examined (Figure 2B). Whereas the proteins extracted from native porcine leaflet specimens initiated 153.7± 27.7x103 cells to transmigrate across the membrane, the decellularization procedure reduced the number of U-937 cells in the LCH to 47.7±5.7x103. The extracts of the human native pulmonary cusps, however, already elicited a smaller amount of 54.4±11.4x103 monocytic cells to transmigrate toward the heart valve proteins. The decellularized human pulmonary valve leaflets again led only 2.1±0.9x103 cells to migrate through the membrane, which was within the range of random migration (range, 1 to 19x103 cells in LCH).
Although the smaller specimens (40 mg; n=7) of the native porcine pulmonary wall revealed a proportionally lower concentration of extracted proteins (40 mg, 0.35±0.04 μg/μL versus 100 mg, 0.83±0.10 μg/μL; P<0.01), the migratory response was similar (40 mg, 22.27±2.57% transmigrated cells versus 100 mg, 24.31±2.38%); 0.36±0.03 μg/μL proteins were extracted from the decellularized porcine conduit wall, but only 5.03±0.14% of U-937 cells were attracted to transmigrate toward the extracts. Similar results were seen when the differently sized specimens of the homograft pulmonary wall were compared (Table). These data indicate that the reduced migration of monocytes truly is caused by the decellularization procedure and does not simply result from the lower concentration of extracted proteins in the LCH.
Weight and Extracted Proteins of Pulmonary Conduit Wall and Cusps and Percentage of Transmigrated Monocytic Cells
Protein Electrophoresis
Electrophoretic separation of homogenized pulmonary cusp and conduit wall tissue demonstrated that the decellularization procedure removes considerable amounts of proteins within the porcine and human heart valves, indicating the effective reduction of potential immunogenic components (Figure 3A and 3B). However, within the examined molecular weight range of 25 to 250 kDa, protein bands were still detectable by SDS-PAGE within the decellularized porcine pulmonary wall tissue. Even the much thinner porcine leaflet tissue revealed residual proteins after the decellularization procedure. In contrast, fewer protein bands were detected in the homogenates of the decellularized human pulmonary wall and leaflet tissue.
Discussion
The durability of conventional glutaraldehyde-treated biological heart valve substitutes is limited because of early calcification or degeneration.9 It is well known that cells or cell membranes within the tissue provoke early calcification of biological heart valve prostheses10,11 whereby a direct connection between specific antibody response and porcine valve tissue calcification has been demonstrated.12 That homograft heart valves elicit a host-dependent immune response against the implanted tissue has been well described.13,14 The concept of decellularization or tissue engineering of heart valves was developed and modified by several groups to circumvent these problems.15–18 Tissue engineering is defined as the regeneration of biological tissue through the use of cells with the aid of supporting structures and/or biomolecules.
Decellularized allogeneic valve prostheses eventually covered with endothelial cells have already been used clinically in humans.4,5 Immunologic evaluations demonstrated strongly decreased antibody response in vivo and satisfactory results in the short term.19–21 Although a decellularization of xenogenous heart valves is also considered to diminish antigenic content,4 early failure and strong inflammatory response against implants were described. The implantation of decellularized porcine heart valves in humans resulted in fibrous sheath formation outside and inside the graft. The histological examination demonstrated a dense neutrophil granulocyte and macrophage reaction around the graft with infiltration of the leaflet tissue,22 which likely represents a standard foreign body–type reaction in which the body fails to remove and consequently tries to encapsulate foreign material. These data raise the suspicion that the decellularized porcine heart valve is not as weakly immunogenic as it is considered to be, and it is highly likely that seeding with endothelial cells might not prevent the immigration of immune competent cells in vivo.
Focusing on the elimination of immunogenic components within a porcine heart valve showed that the detergent-based decellularization procedure revealed no histologically detectable residual cells or cell nuclei. To date, it is unclear to what extent a decellularization procedure needs to remove noncellular integral components of the extracellular matrix like glycosaminoglycans. Because of their ability to interact with cytokines and chemokines, their removal might play an important role in preventing any immune response.23 On the other hand, glycosaminoglycans also appear to play a significant role in tissue elasticity and the prevention of calcification.24 These controversies need to be examined in further studies. Recently, it has been demonstrated in a sheep model that the inflammatory reaction against decellularized heart valves plays a crucial role in valve deterioration.25 The initial events of inflammatory response involve a variety of cell types, including monocytes, and degradation products of extracellular matrix proteins. Monocyte migration is a highly regulated process in which these motile cells sense and respond directionally to chemical gradients, which results in immigration of mononuclear cells at sites of immune response.26
Although U-937 cells represent a transformed monoblastoid cell line, their in vitro behavior parallels that of differentiating mononuclear phagocytes, and their response to chemotactic stimuli is well characterized.27 Using this monocytic cell line for an in vitro migration assay, we could examine the response of human monocytic cells toward decellularized pulmonary heart valve tissue under standardized conditions. We could clearly show that the decellularization of porcine or human heart valves significantly reduces their potential to attract monocytic cells. It is shown that even a much lower protein concentration of native valve specimens still provoked a much higher cell migration than did the extracts of the decellularized heart valve. Thus, it could be proved that the reduced migratory response toward the valve tissue is truly caused by the antigen reduction procedure. It has already been presented that a decellularization process strongly decreases the humoral response in vivo; this study presents a further step in the immunologic evaluation of heart valve substitutes.
However, the porcine pulmonary heart valve treated by the decellularization procedure did not reveal the considered nonantigenic heart valve scaffold. Unexpectedly, only the extracts of the decellularized human pulmonary heart valve did not provoke any monocytic migratory response in vitro. Furthermore, the extracts of the decellularized porcine pulmonary cusps elicited a comparable monocytic migratory response as the untreated cellular homograft leaflets, known to initiate an early host response of inflammatory cells in vivo, predominantly consisting of macrophages.28 Again, the decellularization of human pulmonary heart valves abolished the chemotactic activity of the leaflet tissue.
SDS-PAGE revealed that the antigen reduction procedure effectively removes considerable amounts of proteins within the decellularized heart valve. However, even after the histologically verified effective decellularization procedure, considerable amounts of proteins were still detectable within the xenogenous conduit wall that were not detected within the human equivalent. These remaining proteins may provoke the migratory response seen. Even more surprisingly, we observed that the decellularized porcine heart valve cusps initiated an amount of monocytic cells to transmigrate across the membrane similar to that of the native human cusp, although fewer residual xenogenous proteins were detected by electrophoretic seperation. These data demonstrate that the porcine heart valve treated by an decellularization procedure is not as weakly immunogenic as it is considered to be. The detailed composition of the residual xenogeneic components within the porcine extracellular matrix and the proteins leading to monocyte migration has to be elucidated in further studies currently being carried out in our laboratories.
Because heart valve prostheses have to withstand strong biomechanical stress in vivo from the point of implantation, an enhanced early inflammatory reaction could lead to a significant weakening of the heart valve scaffold and might be the cause of graft rupture.22
In conclusion, we demonstrate for the first time that the migratory response of monocytic cells toward heart valves treated by a decellularization process is significantly different in scaffolds of porcine and human origin. Whereas the decellularized porcine pulmonary valve does not represent a completely nonimmunogenic heart valve scaffold, the decellularization of a human pulmonary heart valve strongly diminishes the migration of monocytic cells toward the valve tissue. Because some of these new biological valve prostheses have already been implanted in humans and others are currently approaching the clinical setting, these findings will have an important impact on further investigations of tissue-engineered heart valves.
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