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Ann Thorac Surg 2004;78:2084-2093
© 2004 The Society of Thoracic Surgeons
a Department of Cardiac Surgery, Rostock, Germany
b Institute for Biomedical Engineering, Rostock, Germany
c Research Center for Cardiac Tissue Replacement, University of Rostock, Rostock, Germany
Accepted for publication March 25, 2004.
* Address reprint requests to Dr Steinhoff, Department of Cardiac Surgery, University of Rostock, Schillingallee 35, D-18057 Rostock, Germany
gustav.steinhoff{at}med.uni-rostock.de
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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Abstract |
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METHODS: Porcine aortic valves were decellularized enzymatically and impregnated with biodegradable poly(hydroxybutyrate) by a stepwise solvent exchange process. Biocompatibility was tested in vitro using cell proliferation and coagulation assays. Proinflammatory activity was assessed in vivo by implantation of matrix/polymer patches in the rabbit aorta. Biomechanic valve properties and fluid dynamics were tested in a pressure/flow-controlled pulse duplicating system. Matrix/polymer hybrid valves were implanted in pulmonary and aortic position in sheep.
RESULTS: Biocompatibility assays indicated that human blood vessel cells survive and proliferate on matrix/polymer hybrid tissue. In vitro activation of cellular and plasmatic coagulation cascades was lower than with uncoated control tissue. After implantation in the rabbit aorta, matrix/polymer hybrid patches healed well, with complete endothelialization, mild leukocyte infiltration, and less calcification than control tissue. Matrix/polymer hybrid tissue had superior tensile strength and suture retention strength, and hybrid valves showed good fluid dynamic performance. The two valves in aortic position performed well, with complete endothelialization and limited inflammatory cell invasion after 12 weeks. Of the two valves in pulmonary position, one failed.
CONCLUSIONS: Matrix/polymer hybrid tissue valves have good biological and biomechanic characteristics and may provide superior replacement valves.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Decellularization
Enzymatic removal of cells without altering the biochemical characteristics of the extracellular matrix by exposure to chemical fixatives was performed as previously described [1]. Briefly, hearts were harvested from porcine cadavers (courtesy of Agricultural Research Center, Dummerstorf, Germany). The aortic root was prepared, washed in phosphate buffered saline (PBS), and incubated in 0.05% trypsin solution for 48 hours at 37°C, followed by 3 washing steps for 1 hour each. For longer storage, the specimens were lyophilized (at –40° C and 0.05 mbar) and rehydrated before further use, but for implantation in sheep the valves were processed immediately. Biocompatibility tests in vitro and in rabbits were performed using human aortic wall tissue, which was obtained during routine coronary artery bypass grafting (CABG) operations and was processed in identical fashion.
Polymer Coating
Biodegradable polymers were supplied by Tepha Inc. (Cambridge, MA). Poly(3-hydroxybutyrate) (P3HB), poly(4-hydroxybutyrate) (P4HB), and poly(3- hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) in powder form were dissolved in chloroform at 50°C according to the chosen concentration. Initially, a dip-coating process was used for fabrication of biomatrix/polymer hybrid tissue: Decellularized and lyophilized tissue was repeatedly immersed in 2% to 6% (w/v) polymer solution followed by solvent evaporation for two weeks until the chloroform content was less than 0.2%. Before further use, the tissue was rehydrated in cell culture medium (in vitro experiments) or phosphate buffered saline (in vivo experiments). When it became evident that this coating may not withstand systemic hemodynamic forces, the protocol was switched to a modified polymer impregnation process: freshly decellularized valve tissue was subjected to a wet dehydration process, replacing water with ethanol. Then, the specimens were immersed in 1% (w/v) polymer solution for 30 minutes, followed by rehydration and wet solvent elimination in PBS.
In Vitro Cell Proliferation
Whether biomatrix/polymer hybrid valve tissue can be repopulated with blood vessel cells, without exhibiting cytotoxicity, was tested in series of in vitro experiments. Tissue samples were prepared as described above, seeded with L929 mouse fibroblasts and incubated under standard cell culture conditions for at least 72 hours. Cell viability was then assessed using the CellTiter96 fluorescent cell proliferation assay (MTS test). Because cellular adhesion, proliferation, metabolic activity, and resistance to toxin are highly dependent on species and cell type, similar tests were performed using a mixed population of human vascular myofibroblasts and endothelial cells, prepared from saphenous vein samples of CABG patients. The lumen was filled with collagenase A containing medium. After incubation for 20 minutes, detached endothelial cells were flushed out and cultivated under standard conditions. The remaining tissue was minced and placed in smooth muscle cell growth medium. Myofibroblasts migrate onto the dish surface, attach, and proliferate. The MTS-tests were performed in decellularized matrix treated with various polymer preparations, but also with pure polymer samples as well as with hydrid tissues following several sterilization and storage protocols such as lyophilization, plasmasterilization, FAD-sterilization, ethylene oxide sterilization, and gamma sterilization (data not shown).
In Vitro Hemocompatibility
Complement and coagulation system activation in response to different hybrid tissue preparations were studied in several in vitro assays. Activation of complement factor C3 was assessed by ELISA for C3a-des-Arg, the stable metabolite of activated C3 (Progen, Heidelberg, Germany), following incubation of human plasma with hybrid tissue samples for 60 minutes. Representative for activation of the plasmatic clotting system, the concentration of the prothrombin fragments F1 and F2 was measured by ELISA (Behring, Marburg, Germany), again after incubation of human plasma with hybrid tissue. Finally, the response of the cellular clotting system to biomatrix/polymer hybrid tissue was studied by measuring platelet factor 4 in human plasma using the AsserachromPF4 assay system.
In Vivo Screening Tests
Intravascular biocompatibility of various hybrid tissue preparations was tested in a rabbit model. Adult New Zealand White rabbits were anesthetized, heparinized, intubated, and ventilated. The abdomen was opened, the abdominal aorta was dissected distal to the renal arteries, clamped, and incised longitudinally. A patch of biomatrix/polymer hybrid tissue measuring approximately 5x3 mm was sutured in place using nonresorbable suture. After 1, 3, or 6 months, the animals were sacrificed and the aortic segment containing the patch was explanted and prepared for histology. Sections were stained with H&E or antibodies for immunohistology, and examined by light microscopy. A scoring system was designed to facilitate comparison of histologic findings. The following histologic characteristics were studied: endothelialization of the luminal patch surface, intima proliferation, inflammatory infiltration, calcification, cellular migration into the patch material, thrombus formation, and formation of a neo-elastica interna (Table 1 ).
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Large Animal Implantation
Once the optimal biomatrix/polymer tissue composition had been chosen, four porcine hybrid valves were implanted in sheep by Experimental Surgical Services (ESS), University of Minnesota. Following approval by the local regulatory bodies, two valves were implanted to replace the native pulmonary root, and three valves were implanted as freestanding aortic root replacement with coronary reimplantation. One animal died while undergoing aortic valve surgery and was excluded from the analysis. Three months after implantation, the animals underwent echocardiography and cardiac catheterization and were sacrificed. Morphologic analysis of the hybrid valve was carried out by the local pathologist before the valve tissue was divided in three parts and prepared for further analysis by light microscopy, immunohistology, and electron microscopy.
Histology
Hematoxylin & eosin, Mason's trichrome, and Van Gieson staining of formalin-fixed paraffin-embedded tissue was performed in standard fashion. For immunohistology, frozen sections were prepared from cryopreserved tissue and incubated with monoclonal mouse antihuman CD31 antibody (Dako, clone Nr. JC70A) or monoclonal mouse antihuman smooth muscle actin antibody (Dako, clone Nr. 1A4), followed by detection with peroxidase-conjugated goat antimouse IgG secondary antibody (Dako). For analysis of surface morphology and ultrastructure, specimens were prepared for scanning electron microscopy and viewed using a Philips XL30ESEM (FEI, Hillsboro, OR) electron microscope.
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Results |
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The first valve implanted in pulmonary position was severely obliterated, with severe diffuse pio-granulomatous valvular endocarditis, chronic lympho-histocytic and necrotizing bioprosthetic periarteritis, and intimal fibrous-hyperplasia. Although microbiology at the time of sacrifice was negative, bacterial endocarditis was probably the cause (Fig 5A). The second valve was in excellent condition, with near-normal gross morphology (Fig 5B). On histology, there was complete endothelial cell lining and moderate multifocal granulomatous inflammation limited to the leaflet hinge point (data not shown).
Particular attention was paid to the two valves implanted in aortic position. At the time of sacrifice, both animals were in good condition without clinical signs of valve dysfunction. Gross morphology and representative photomicrographs are shown in Figure 6. The pathologist described that there were focal fibrinous deposits on the leaflets. The leaflets contained homogeneous eosinophilic bundle of collagens, expanded by moderate multifocal granulomatous inflammation (epitheloid macrophages and giant multinucleated cells). As seen in Figure 6B, there was some intimal thickening particularly of the luminal surface of the otherwise delicate leaflets. By immunohistology, complete endothelial cell lining of the leaflets and the conduit wall was found. There was smooth muscle cell migration into the media of the leaflets (Fig 7), but very little cellular infiltration of the conduit wall (Fig 6E). By scanning electron microscopy, the luminal surface of the leaflets in aortic position showed a smooth texture without apparent interruptions of the intimal integrity (Fig 8).
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Comment |
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Due to the multiple shortcomings of conventional prostheses, heart valve tissue engineering strategies have attracted considerable attention. The initial approach was based on the fabrication of the entire valve scaffold from biodegradable polymers, followed by in vitro seeding with autologous cells and conditioning under simulated in vivo conditions before implantation [1–3]. Early results were promising, but it soon became clear that the complex three-dimensional structure of the native valve can hardly be achieved with current techniques, and the structural and mechanical properties of the various polymers are not ideal. Furthermore, in vitro seeding and conditioning with cells of the future recipient is a time-consuming process that would require sophisticated laboratory equipment in the clinical setting. In addition, it remains unclear whether the cells actually adhere to the scaffold after implantation, and there is evidence that the cells found on in vitro-seeded valves months after implantation are in fact cells that have colonized the valve postimplantation, independent from the preseeding process. More recently, natural xenogenic or allogenic heart valve tissue has been propagated as a scaffold for heart valve tissue engineering. In contrast to aldehyde-fixed tissue, enzymatically decellularized extracellular matrix without tanning-induced crosslinks possesses epitopes for cellular adhesion receptors, facilitating repopulation with tissue-specific celltypes but also inflammatory cells [6, 7]. Nonautologous matrix constituents such as collagen, elastin, and proteoglycans have little antigenicity, given that cellular components are entirely removed [8, 9] It has recently been shown that mismatch of HLA-DR and ABO antigens on endothelial cells in unmodified valve allografts is associated with accelerated valve failure [10, 11]. To avoid the immunologic response to graft cells, we have developed a decellularization process that removes cells and cellular debris from the heart valve scaffold by enzymatic digestion, without the use of matrix-altering tanning procedures. Thus, the biological properties of the extracellular matrix components are preserved, and the decellularized valve can be repopulated by cells derived from the recipient organism. In an earlier series of experiments, we reseeded such decellularized valve scaffolds with autologous blood vessel cells in vitro and implanted those in pulmonary position in sheep [4]. Three months after implantation, good valve function and complete histologic restitution of valve tissue, as well as a confluent endothelial surface were found. However, aggressive enzymatic decellularization inevitably weakens the valve tissue, so that the mechanical properties do not allow for implantation in the high pressure system. Therefore, we sought to combine the advantageous properties of the native extracellular matrix scaffold with those of an artificial polymer and demonstrated that polymer-treated valve scaffolds exhibit improved resistance to hemodynamic forces [5]. These composite heart valves can now be implanted in vivo, and will be repopulated by recipient cells. The polymer coating will then be degraded, while cells migrate into the extracellular matrix scaffold, restore the natural tissue structure of the native heart valve, and initiate the physiologic turnover of extracellular matrix components. Once these processes have been completed, the neo-valve should have biological and mechanical characteristics identical to those of the native valve.
The coating process also serves to attenuate the pro-coagulatory activity of bare matrix components. It is well established that platelet activation occurs when platelets come in contact with collagen. Platelets directly adhere by binding of platelet collagen receptor to integrins on collagen, and the intrinsic clotting cascade is initiated when prekallikrein, kininogen, factor XI and factor XII are exposed to collagen. In in vitro tests screening tests we found that activation of both the cellular and the plasmatic coagulation system is indeed attenuated by polymer coating of decellularized matrix, while there was no apparent change in the extent of complement activation.
The use of uncoated decellularized matrix with or without in vitro autologous preseeding for heart valve replacement remains controversial. Recently, Leyh and colleagues [12] reported that decellularized porcine pulmonary valves implanted in pulmonary position in growing sheep functioned well for up to 24 weeks and exhibited reconstitution of viable valve tissue and only mild calcification. On the other hand, allogenic sheep valves prepared in the same manner calcified rapidly, associated with structural and functional deterioration. In a similar model, the same group observed that in vitro repopulation with autologous cells before implantation led to severe leaflet degeneration in vivo, while valves that were implanted unseeded had clearly superior morphologic and functional characteristics [13]. In another study, however, they observed progressive degeneration of xenogenic decellularized aortic tissue when implanted subcutaneously in rats, and concluded that some recellularization process may be necessary before implantation [14].
Decellularized valves are already commercially available. Preclinical large animal testing in the xenogenic and allogenic setting was very promising, with good hemodynamic function, morphologic reconstitution, and little calcification for almost 1 year [15–17]. The initial enthusiasm has been muted, however, by reports of early xenograft failure in humans [18, 19]. Decellularized homografts appear to cause less problems in humans, but the actual long-term benefit remains to be determined [20, 21]
In conclusion, we believe that decellularized heart valve matrix holds great potential for creation of viable, long-lasting replacement valves, and that many of the problems in the xenogenic setting, including impaired biomechanics and residual antigenicity can be overcome by pretreatment with biodegradable polymers. However, more extensive long-term studies in large animals are clearly needed before clinical use can be considered.
Discussion
DR SCOTT M. BRADLEY (Charleston, SC): In terms of the final biopolymer you arrived at—I think it was a combination of two polymers—can you give us an idea of how long that takes to degrade in vivo.
DR STAMM: The lifetime in vivo of P3 and P4HB is different, the half-lives are different. For P3HB, I believe they are in the magnitude of several months, if not years; P4HB degrades much faster. About the overall total degradation time or half-life of the polymer combinations, nothing is known.
DR ANTONIO CORNO (Lausanne, Switzerland): The composite valves that you prepared are exposed to different regimen in pulmonary artery position regarding the pressure and the oxygen saturation. Did you detect any difference to the response of this valve?
DR STAMM: The valve in pulmonary position that survived the procedure more or less intact had a complete endothelial lining. We also found some smooth muscle cells in the leaflets, but we were not able to detect a significant difference in terms of histologic appearance between both implantation sites, aortic or pulmonary.
DR HENRY L. WALTERS III (Detroit, MI): Did you do any scanning electron micrography on the autopsy specimens of the valves that you put in circulation to determine the uniformity of endothelialization of the leaflets?
DR STAMM: Do you mean like the image that I showed of the leaflet in aortic position?
DR WALTERS: That looked like a cross-section light micrograph to me. But you had shown some scanning electron micrograph specimens of your combination matrices, and I only saw light micrographs of a cross-section of the autopsy specimens after they had been in circulation. I thought the endothelialization was one of the remarkable findings, and I just wondered how uniform it is and if you have done any studies on the cells to assess their function.
DR STAMM: As yet we only have cross-sectional images of both conventional staining and immunohistology, but the endothelial lining is complete in all the sections that we've looked at. We have also looked at the entire surface by scanning electron microscopy and again observed a complete cell lining.
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Acknowledgments |
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References |
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This article has been cited by other articles:
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  D. Schmidt, U. A Stock, and S. P Hoerstrup Tissue engineering of heart valves using decellularized xenogeneic or polymeric starter matrices Phil Trans R Soc B, August 29, 2007; 362(1484): 1505 - 1512. [Abstract] [Full Text] [PDF]
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 A. Vincentelli, F. Juthier, and B. Jude Reply to the editor. J. Thorac. Cardiovasc. Surg., September 1, 2006; 132(3): 737 - 737. [Full Text] [PDF]
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C. Stamm and G. Steinhoff When less is more: Go slowly when repopulating a decellularized valve in vivo! J. Thorac. Cardiovasc. Surg., September 1, 2006; 132(3): 735 - 737. [Full Text] [PDF]
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 I. Vesely Heart Valve Tissue Engineering Circ. Res., October 14, 2005; 97(8): 743 - 755. [Abstract] [Full Text] [PDF]
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