Ann Thorac Surg 2010;89:259-263. doi:10.1016/j.athoracsur.2009.06.048
© 2010 The Society of Thoracic Surgeons
New Technology
Percutaneous Tissue-Engineered Pulmonary Valved Stent Implantation
Georg Lutter, MD, PhDa,*,*,
Anja Metzner, PhDa,*,
Thomas Jahnke, MD, PhDa,
René Bombien, MDa,
Jessica Boldt, MSa,
Kenji Iino, MDa,
Jochen Cremer, MD, PhDa,
Ulrich A. Stock, MD, PhDb
a Departments of Cardiovascular Surgery and Radiology, Christian-Albrechts-University of Kiel, School of Medicine, Kiel, Germany
b Department of Thoracic, Cardiac and Vascular Surgery, University Hospital Tuebingen, Eberhard Karls University, Tuebingen, Germany
Accepted for publication June 12, 2009.
* Address correspondence to Dr Lutter, Department of Cardiovascular Surgery, Christian-Albrechts-University of Kiel, School of Medicine, Arnold-Heller-Str. 7, Kiel, 24105, Germany (Email: lutter{at}kielheart.uni-kiel.de).
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Abstract
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Purpose: The purpose of this study was to evaluate the feasibility of percutaneously implanted tissue-engineered valved stents in the ovine pulmonary valve position.
Description: Porcine pulmonary heart valves and small intestinal submucosa were obtained from a slaughterhouse, and the intestinal submucosa used to cover the inside of the porcine pulmonary valved stents. Endothelial cells and autologous myofibroblasts were obtained from carotid artery segments of juvenile sheep. After myofibroblast seeding, constructs were placed in a dynamic bioreactor system and were cultured for 16 days. After Endothelial cell seeding, the tissue-engineered valved stents were deployed into the pulmonary valve annular site. Angiography was performed at implantation and explantation (4 weeks). Constructs were analyzed macroscopically and microscopically.
Evaluation: Orthotopic positioning of the stents (n = 3) at the time of implantation and explantation, as well as normal valve function, was observed through angiography. Gross morphology confirmed excellent opening and closing of all leaflets. Strong expression of 
-smooth muscle actin in neointerstitial cells and of von-Willebrand-Factor in endothelial cells was revealed by immunocytochemistry.
Conclusions: This study demonstrates successful merging of two novel technologies: (1) percutaneous valved stent implantation and (2) tissue engineering of autologous heart valves.
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Introduction
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Heart valve replacement is the most common surgical therapy for end-stage valvular heart disease. Recently, use of transfemoral access to replace pulmonary heart valves with glutaraldehyde fixed bovine jugular vein valves has been clinically introduced [1]. Unfortunately, these valves are prone to degeneration and long-term failure. Clinical implantation of decellularized xenograft valves resulted in early failure, potentially caused by preserved matrix immunogenicity [2]. Autologous tissue engineering might offer an attractive method to overcome the previously mentioned limitations [3].
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Technology
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For this study pulmonary heart valves were used (n = 3). Porcine pulmonary heart valves and small intestinal submucosa were obtained from a local slaughterhouse. After immediate dissection and antimicrobial incubation, valves were trimmed (removal of sinus and most of the muscular ring) to fit into the deployment catheter. The porcine valve was decellularized with 0.02% trypsin and 0.02% trypsin ethylenediamine tetra acetic acid (Biochrome AG, Berlin, Germany), and was sutured into a self-expanding nitinol stent (Nitinol Devices & Components Inc, Fremont, CA) as previously described [4].
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Technique
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A 4-cm long segment of the recipient ovine carotid artery was removed under sterile conditions. Endothelial cells were obtained using 0.2% collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) and cultured on a 1% gelatine-coated (gelatine type A [Sigma Aldrich Chemie GmbH, Munich, Germany]) 24-well plate (Nunc GmbH, Wiesbaden, Germany) in a cell culture medium (M199-medium, 1% penicillin–streptomycin, 10% fetal calve serum, and recombinant human fibroblast [Peprotech, Hamburg, Germany]).
The ovine vessel wall segments were minced into 1 mm2 pieces, distributed on a 6-well plate and cultured in cell culture medium. After moving the myofibroblasts onto culture dishes (5 to 9 days after harvest), cells were cultured in gelatine pre-coated flasks. Both cell populations were incubated at 37°C and 5% CO2 and serially passaged with trypsin ethylenediamine tetra acetic acid. A scaffold was seeded with a mix of fibroblasts and smooth muscle cells (myofibroblasts) isolated from the carotid artery. These seeded valved stents were kept in a dynamic bioreactor for 16 days and finally seeded with endothelial cells for 4 days.
For implantation, the construct was folded carefully and inserted into a modified commercially available delivery system [5]. Through a 22F sheath positioned in the right femoral vein, the 20F application device was inserted and the autologous valved stent was successfully positioned on top of the native pulmonary valve under fluoroscopic control as previously described [5]. Four weeks after implantation, animals underwent re-angiography with subsequent stent explantation followed by gross morphology examination and processing for histology and immunohistochemistry.
The explanted valved stent was subsequently fixed in 4% formalin. Each leaflet was carefully removed from the stent and the three leaflets were prepared as previously described [4]. For general morphological analysis, serial sections were stained with hematoxylin and eosin stain and Russel-Movat's pentachrome stain. To confirm endothelial cells on tissue surface, von Willebrand factor and endothelin staining (platelet/endothelial cell adhesion molecule-1) were conducted. To identify myofibroblasts, -actin related antigen staining was performed. Sections were analyzed and documented using a brightfield light microscopy (Axiovert 135 [Zeiss, Jena, Germany]).
Angiography
The angiography was performed 30 minutes and 4 weeks after successful implantation, as previously described [5].
Echocardiography
Echocardiographic studies were conducted after 2 weeks, as described elsewhere [5].
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Clinical Experience
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This procedure was performed in three juvenile sheep weighing 40.5 ± 0.9 kg. The mean diameter of the native pulmonary annuli was 18.3 ± 1.5 mm (range, 17.0 to 20.0 mm) as revealed by angiography. Implanted valved stents with a maximal outer diameter between 22.1 and 24.2 mm were used. Mean duration of the procedure from insertion of the application device through the sheath to deployment was 56.9 ± 10.2 seconds on average.
Angiography and Echocardiography
Angiographic observation indicated correct orthotopic positioning of each valved stent at the time of implantation and after 1-month follow-up (Fig 1). Angiography revealed no regurgitations after 4 weeks and showed competent valved stents in all three surviving sheep. A 1-month angiography and echocardiography demonstrated good opening and closing of the implanted valves in the normal functioning heart.
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Fig 1. Correct positioning of a tissue-engineered autologous valved stent in the pulmonary position in an ovine model (A) at the time of implantation and (B) at the 4-week follow-up.
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Hemodynamic Measurements
During the deployment procedure, rhythm disturbances occurred in all animals. The peak-to-peak gradient across the valved stents was initially 2.3 ± 1.2 mm Hg, and 4 ± 5 mm Hg at follow-up (p = not significant). The hemodynamic data are shown in Table 1.
Macroscopic Analysis
Macroscopic analysis of the pulmonary heart valve stent demonstrated optimal orthotopic positioning (Fig 2A). Macroscopic analysis of the leaflets showed smooth surfaces with no thrombus formation (Fig 2B). No paravalvular defects were observed. The valved stents were pliable and the leaflets were thin without indurations. A slight fibrous overgrowth was limited to the outflow portions of the valved stents. Cardiac structures were unscathed. No macroscopic damage was noted.
Histology
Hematoxylin and eosin staining made general morphology of the leaflet visible (Fig 3A).
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Fig 3. (A) Hematoxylin and eosin staining (original magnification x200) and (B) Russel-Movat's pentachrome staining (original magnification x200) of tissue-engineered heart valve 4 weeks after implantation (bar represents 100 µm).
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The Russel-Movat's pentachrome staining (Fig 3B) made the physiologic valve structure visible with preservation of arterialis, fibrosa, spongiosa and ventricularis, and no apparent disruptions of the leaflet histoarchitecture. A confluent endothelial cell lining of the leaflets (identified by the presence of factor VIII-related antigen and endothelin antibody [platelet/endothelial cell adhesion molecule]) displayed typical cobblestone morphology (Figs 4A and 4B). Immunohistochemistry demonstrated strong expression of -smooth muscle actin and an ingrowth into the leaflets (Fig 5).
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Fig 4. (A) Endothelial staining with platelet/endothelial cell adhesion molecule-1 (original magnification x200; bar represents 100 µm) and (B) Immunofluorescence von-Willebrand-Factor staining (original magnification x200; bar represents 100 µm).
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Statistical Analysis
Values are presented as mean ± standard deviation. Data were analyzed based on the paired t test to compare related data for non-normally distributed data using the SPSS 10.1 software (SPSS Inc, Chicago, IL). The p values less than 0.05 were considered statistically significant.
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Comment
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Right ventricular outflow tract (RVOT) reconstruction with valved conduits in infancy and childhood leads to reintervention for pulmonary regurgitation and stenosis in later life [6]. The transannular patch of the RV outflow tract may aggravate pulmonary regurgitation with deleterious effects on long-term outcome, such as RV enlargement and dysfunction, reduced exercise capacity, malignant arrhythmia, and sudden cardiac death [7]. The Contegra conduit induces a neointimal proliferation at the level of the pulmonary anastomosis. This leads to a high incidence of severe stenosis at intermediate-term follow-up [8]. Accordingly, percutaneous pulmonary valve implantation techniques were developed to offer these young patients with dysfunctional conduits a less invasive treatment option. After extensive in vivo evaluation, Bonhoeffer and colleagues [9, 10] introduced the concept of endovascular pulmonary valve application clinically.
This concept represents a tremendous benefit for the patients as it avoids repeat operations; the currently used bovine jugular vein valves are glutaraldehyde-fixed xenogeneic heart valves prone to degeneration and will eventually require reinterventions. Tissue engineering might offer a potential pathway to overcome the present limitations of heart valve substitutes in general [3].
Tissue-engineered valves will have the unique characteristics of viability and ability to grow and remodel. In this animal study we implanted the valves percutaneously, which is more evident for the patient than a conventional technique. We combined percutaneous valved stent implantation and tissue engineering of autologous heart valve cells on decellularized porcine xenografts. In the analyzed sheep we have now shown that the angiography and gross morphology in the analyzed tissue-engineered valved stents demonstrated good opening and closing of the tissue valves after 4 weeks. Orthotopic positioning of the stents at the time of implantation and explantation was confirmed. Furthermore, immunocytochemistry revealed strong expression of -smooth muscle actin in neo-interstitial cells and of von-Willebrand-factor in endothelial cells.
Tissue engineering might bear promising solutions to surmount the limitations of biological heart valves, which are prone to degeneration [11]. Easily available porcine heart valves were used after their decellularization process. They served as an optimal scaffold for the new valved stent. It was laminated with autologous ovine myofibroblasts and endothelial cells that were harvested from ovine carotid arteries. This concept was used to avoid any immunological and inflammatory reactions that were not observed macroscopically or microscopically in this study.
After open heart surgery, the Hannover group has shown tissue-engineered valves with autologous cells that might have the potential to remodel and grow according to the somatic growth of children [12]. They isolated mononuclear cells from children's peripheral blood, seeded these cells on a decellularized porcine pulmonary valve, and placed it in a bioreactor for 21 days. Then the valve was implanted into the children. The use of autologous cells was also not connected with immunoreactions or signs of inflammation. This is one of the advantages of using autologous cells. The concept of tissue engineering is based on the usage of decellularized biological matrices; therefore the removal of dissimilar cellular components might reduce immunological reactions.
The Berlin group analyzed the difference between in vitro endothelial cell seeding valves and nonseeding valves for 3 and 6 months in a sheep model. They demonstrated that it is not necessary to use in vitro seeding, because there was no difference between the two groups after 6 months. They also discussed the advantage of in vitro endothelial cell seeding of tissue-engineered heart valves that might protect against early platelet aggregation [13]. Andrews and Berndt [14] demonstrated the potential stimulation of blood vessel wall injury to create activated platelet aggregates or thrombus acceleration due to glycoprotein availability in collagen. These adhered, activated platelets might also interact with inflammatory leukocytes and facilitate platelet leukocyte endothelial cell adhesion.
Therefore we used cells from the carotid segment of a sheep and implanted the tissue-engineered heart valve stent into the same sheep. After 4 weeks the stent was explanted and the leaflets were processed for histology, which showed no inflammation and in-growth of the cells into the leaflet. However, we have also found some calcification (unpublished data, 2008).
A variety of limitations remain to be addressed. In addition to evaluation of the optimal cell source for seeding of the constructs [15], the potential immunologic barrier of porcine extracellular matrix tissue in human subjects needs further investigation [16]. An important and crucial limitation of mid-term to long-term tissue engineering in general is an immanent infection risk. Children must be reoperated on several times because of the valved stent does not grow. The cells should be from either bone marrow or, better yet, from peripheral blood-derived cells for a pure population of endothelial cells and myofibroblasts for tissue engineering.
The soft nitinol stent adaptability to the surrounding structures could be beneficial for physiologic blood flow in the proximal great arteries compared with steel or platinum stents. This study indicated that the right ventricular and pulmonary artery pressures were increased compared with preoperative data, whereas no significant transvalvular gradients were revealed in our tissue-engineered valves. During the implantation, rhythm disturbances were observed without affecting the normal heart function (Table 1). Good right and left ventricular heart function was retained after 4 weeks.
The present study demonstrates successful application of tissue-engineered pulmonary heart valves. Although the data are very encouraging, there are still a number of unanswered questions regarding tissue engineering. Therefore, more animals will be analyzed. Two more animals are still alive and follow-up assessment will be performed monthly for determination of their long-term function and durability.
In conclusion, this study demonstrates successful simultaneous application of two novel technologies: (1) percutaneous valved stent implantation and (2) tissue engineering of autologous heart valves in an animal model for 4 weeks.
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Disclosures and Freedom of Investigation
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The authors of this article had full control of the design of the study, methods used, outcome measurements, analysis of data, and production of the written report. The authors had no financial relationship with any companies.
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Acknowledgments
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This project is supported by the German Research Foundation, Bonn, Germany (LU663/7-2 and STO359/4-3). We gratefully thank Christine Haß, Marion Krüger and Ilka Degenkolbe for their excellent technical assistance.
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Footnotes
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Disclaimer The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article.
* These authors contributed equally to this work. 
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References
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- Shinoka T, Breuer CK, Tanel RE, et al. Tissue engineering of heart valves: valve replacement study in a lamb model Ann Thorac Surg 1995;60:S513-S516.[Medline]
- Simon P, Kasimir MT, Seebacher G, et al. Early failure of the tissue engineered porcine heart valve SYNERGRAFT in pediatric patients Eur J Cardiothorac Surg 2003;23:1002-1006.[Abstract/Free Full Text]
- Nerem RM. Cellular engineering Ann Biomed Eng 1991;19:529-545.[Medline]
- Stock UA, Degenkolbe I, Attmann T, Schenke-Layland K, Freitag S, Lutter G. Prevention of device-related tissue damage during percutaneous deployment of tissue-engineered heart valves J Thorac Cardiovasc Surg 2006;131:1323-1330.[Abstract/Free Full Text]
- Attmann T, Quaden R, Jahnke T, et al. Percutaneous pulmonary valve replacement: 3-month evaluation of self-expanding valved stents Ann Thorac Surg 2006;82:708-714.[Abstract/Free Full Text]
- Frigiola A, Redington AN, Cullen S, Vogel M. Pulmonary regurgitation is an important determinant of right ventricular contractile dysfunction in patients with surgically repaired tetralogy of Fallot Circulation 2004;110(Suppl):II153-II157.[Medline]
- Abd El Rahman MY, Abdul-Khaliq H, Vogel M, et al. Relation between right ventricular enlargement, QRS duration, and right ventricular function in patients with tetralogy of Fallot and pulmonary regurgitation after surgical repair Heart 2000;84:416-420.[Abstract/Free Full Text]
- Meyns B, Van Garsse L, Boshoff D, et al. The Contegra conduit in the right ventricular outflow tract induces supravalvular stenosis J Thorac Cardiovasc Surg 2004;128:834-840.[Abstract/Free Full Text]
- Bonhoeffer P, Boudjemline Y, Saliba Z, et al. Transcatheter implantation of a bovine valve in pulmonary position Circulation 2000;102:813-816.[Abstract/Free Full Text]
- Bonhoeffer P, Boudjemline Y, Saliba Z, et al. Percutaneous replacement of pulmonary valve in a right-ventricle to pulmonary-arteryprosthetic conduit with valve dysfunction Lancet 2000;356:1403-1405.[Medline]
- Vesely I. Heart valve tissue engineering Circ Res 2005;97:743-755.[Abstract/Free Full Text]
- Cebotari S, Lichtenberg A, Tudorache I, et al. Clinical application of tissue engineered human heart valves using autologous progenitor cells Circulation 2006;114(Suppl I):I-132-I-137.[Abstract/Free Full Text]
- Dohmen PM, da Costa F, Yoshi S, et al. Histological evaluation of tissue-engineered heart valves implanted in the juvenile sheep model: is there a need for in-vitro seeding? J Heart Valve Dis 2006;15:823-829.[Medline]
- Andrews RK, Berndt MC. Platelet physiology and thrombosis Thromb Res 2004;114:447-453.[Medline]
- Schenke-Layland K, Riemann I, Opitz F, et al. Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves Matrix Biol 2004;23:113-125.[Medline]
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Abstract
Introduction
