Asian Cardiovasc Thorac Ann 2002;10:25-30
© 2002 Asia Publishing EXchange Pte Ltd
Tissue Engineering of an Auto-Xenograft Pulmonary Heart Valve
Pascal M Dohmen, MD,
Shigeyuki Ozaki, PhD1,
Erik Verbeken, PhD2,
Jessa Yperman,
Willem Flameng, PhD1,
Wolfgang F Konertz, PhD
Department of Cardiovascular Surgery University Hospital Charité Humboldt-University Berlin Berlin, Germany
1 Center for Experimental Surgery and Anesthesiology
2 Department of Pathology Catholic University Louvain Louvain, Belgium
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Pascal M Dohmen, MD Tel: 49 30 450 522 092 Fax: 49 30 450 522 921 email: pascal.dohmen{at}charite.de Department of Cardiovascular Surgery, University Hospital Charité, Humboldt-University Berlin, Schumannstraße 20/21, Berlin D-10098, Germany.
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ABSTRACT
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To improve the durability of stentless valves without losing their excellent hemodynamic function, a new-generation auto-xenograft was developed and evaluated. A piece of vein was harvested from 3 juvenile sheep 6 weeks before implantation of the valve. Endothelial cells from the vein material were cultivated and used to reendothelialize a decellularized porcine pulmonary valve. The tissue-engineered valve was implanted into the right ventricular outflow tract of the juvenile sheep. It was explanted after 100 days and assessed macroscopically as well as by x-ray, light microscopy (hematoxylin and eosin staining and von Kossa staining), and scanning electron microscopy. Calcium content of the cusps was determined quantitatively by atomic absorption spectrometry. The sheep implanted with the valve recovered quickly without any problems during the observation period. X-ray examination of the 3 explanted valves showed no cusp calcification, which was confirmed by histological study. Atomic absorption spectrometry showed low tissue calcium content. A clinical safety and feasibility trial with an allograft valve prepared the same way showed excellent short-term results in 6 patients.
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INTRODUCTION
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Until now, no valve prostheses are available that offer long-term durability, excellent hemodynamic function, and regeneration potential. Mechanical valve prostheses have the disadvantage of lifelong anticoagulation with the inherent comorbidity,1 while biological prostheses are limited by the lack of durability caused mainly by tissue calcification, especially in young adults.2,3 Allografts, too, tend to calcify, especially in young patients.4 A better treatment may be the Ross operation, in which the diseased aortic valve is replaced with an autologous pulmonary valve. With the advent of tissue engineering, it is now possible to produce living tissues made from an acellular matrix seeded with autologous endothelial cells.5 Such grafts should have the potential to grow and regenerate. The aim of this study was to evaluate the so-called auto-xenograft in vivo.
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MATERIALS AND METHODS
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All experiments were performed in accordance with the "Principles of Laboratory Animal Care" prepared by the National Society of Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institute of Health (NIH publication 85-23, revised 1985). This study was approved by the ethics committee of the Catholic University Louvain.
PREPARATION OF VEIN MATERIAL AND ENDOTHELIAL CELL HARVESTING
The vein material was harvested from 2-month-old juvenile sheep, which were fasted for 24 to 36 hours before the induction of general anesthesia. All animals were premedicated with ketamine (Ketalar; Parke-Davis, Zavetem, Belgium) 10 to 20 mg•kg-1 intramuscularly. Albipen LA (Mycofarm, Brussels, Belgium) 15 mg•kg-1 was administered intramuscularly for antibiotic prophylaxis. Anesthesia was induced with 4% halothane oxygen mixture. After intubation with a Portex tube (size 7 to 10), the sheep was ventilated with an Engtröm 200 respirator (Helsinki, Finland). All ventilation parameters were adjusted to keep blood gas levels within the normal range. With the animal placed in a right lateral decubitus position, a peripheral intravenous line was placed in the left lower limb, and arterial blood pressure was measured by ear artery cannulation. A 3-lead electrocardiogram was used to monitor heart rate and rhythm. Under sterile conditions, a skin incision was made at the leg or the neck, and the great saphenous or jugular vein was prepared over a length of 15 to 20 cm and cannulated with 2-mm cannulas on both sides. After ligation of the side branches, the vein was rinsed and stored in DelBecco's Modified Eagle's Medium (DMEM) (Sigma Chemical, St. Louis, MO, USA) with an antibiotic solution (penicillin 100 U•mL-1, streptomycin 100 µg•mL-1, and ampho-tericin B 250 ng•mL-1; Sigma Chemical, St. Louis, MO, USA). It was then transported to the cell culture laboratory, where endothelial cells were separated from it with 0.1% Collagenase P (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA). The vein with the collagenase was placed in a humidified incubator at 37°C, 5% CO2, and 98% air saturation for 15 minutes. The separated endothelial cells were cultured for 4 weeks in DMEM with 20% autologous sheep serum and 5 µg•mL-1 basic fibroblast growth factor (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA). The medium was changed every 2 days, and endothelial cell growth was evaluated by daily microscopic examination.
PREPARATION OF ACELLULAR MATRIX AND AUTO-XENOGRAFT
Porcine pulmonary valves were harvested at the slaughterhouse and transported to the laboratory in Hanks solution (Sigma Chemical, St. Louis, MO, USA) with the same antibiotic solution mentioned earlier. The pulmonary valves were macroscopically inspected and underwent bacteriologic control. After preparation and control of the leaflets for fenestration, the valves were sized and a competence test conducted.6 They were then stored in Hanks solution at 4°C for no longer than 7 days. Prior to seeding, the porcine tissue was chemically decellularized (Figure 1
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Figure 1. A representative part of the pulmonary wall of a porcine pulmonary valve that was chemically decellularized. An intact collagen matrix can be seen (hematoxylin and eosin stain, original magnification x25).
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After 3 hours' immersion in fibronectin (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA) to increase their binding potential, the endothelial cells were seeded on the inside of the acellular matrix in a 2-dimensional way for a period of 4 hours. The auto-xenografts were then kept in a humidified incubator at 37°C and 5% CO2 for 7 days.
IMPLANTATION OF AUTO-XENOGRAFT IN JUVENILE SHEEP
Anesthesia was induced as described above. A left minithoracotomy was performed at the 2nd intercostal space. Before opening the pericardium, 3 mg•kg-1 heparin (Novo, Bagsvaerd, Denmark) and 100 mg lidocaine (Xylocard; Astra, Södertälje, Sweden) were administered intravenously. The pericardium was incised vertically to expose the heart. The main pulmonary artery (PA) was isolated, and pursestring sutures of 4/0 polypropylene were placed on the distal part of the PA and 3/0 polypropylene on the right atrium. The PA was cannulated for arterial perfusion using a 24F cannula, and the right atrium was cannulated for venous return using a 34F cannula. A 54-mL Medos VAD System (Medos AG, Aachen, Germany) was connected to both cannulas and adjusted to maintain a mean systemic blood pressure of 50 to 60 mm Hg. The PA was transected after both sides were clamped. The auto-xenograft was interposed using continuous 4/0 polypropylene sutures for proximal and distal anastomoses. Great care was taken not to touch the graft on the luminal surface. After deairing, the clamps were released and ventricular support discontinued. The native pulmonary valve subsequently was made incompetent by tearing the leaflets. The thoracotomy was closed in layers after insertion of a chest drain. The chest drain was removed 2 hours postoperatively.
EXPLANTATION AND ANALYSIS
The auto-xenografts (n = 3) were explanted after 3 months. The left minithoracotomy was reopened and the heart dissected free. Heparin 3 mg•kg-1 was administered, and after exsanguination the valve was excised together with the proximal and distal parts of the sheep's PA. The explanted prostheses were grossly inspected, and color photographs were taken. Special attention was paid to cusp retraction, leaflet fenestration, or thrombus formation on any parts of the valve. Each graft was longitudinally transected through the commissures, including a part of the sheep's PA on both sides.
The valves were examined for calcification by x-ray (face, profile) performed under mammography conditions. For quantitative calcium determination, the anterior pulmonary cusps were divided into 3 parts: commissural area, middle part, and free edge. After lyophilization, the tissue was pulverized and desiccated to constant weight in an oven. Hydrolysates were made in 0.5 mL of 2M hydrochloric acid. Calcium content was measured by flame atomic absorption spectrometry.
To prepare for light microscopy, a longitudinal section of the specimen through the middle of the left pulmonary cusp was embedded in paraffin, and sections 4-µm thick were routinely stained with hematoxylin and eosin as well as von Kossa. For surface analysis by scanning electron microscopy, the posterior pulmonary cusp was also divided into a commissural part, a middle part, and the free edge, including the native pulmonary trunk on both sides. Tissue specimens were dried by the critical point method and covered with gold. They were examined with a Philips Xl-40 scanning electron microscope (Eindhoven, The Netherlands).
Immunohistochemical staining was performed to detect the presence of factor VIII-related antigen in the endothelial-like cells of the auto-xenograft. Several representative parts of the pulmonary wall and leaflets were investigated.
CLINICAL STUDY
At the University Hospital Charité of Humboldt-University Berlin between May and December 2000, 6 patients were given a tissue-engineered valve prosthesis after obtaining institutional review board approval and informed consent. The graft was used to reconstruct the right ventricular outflow tract during aortic valve replacement according to the Ross technique. The valve was produced using the same procedure as in the animal study, the only difference being the use of a commercially available pulmonary valve allograft, which was decellularized and then seeded with the patient's own endothelial cells. Echocardiography and magnetic resonance imaging were performed at discharge and after 3 months.
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RESULTS
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After 4 weeks' culture, the mean number of endothelial cells available was 58.35 x 10-6 (range, 25.74 x 10-6 to 108.64 x 10-6) for seeding the acellular porcine pulmonary valves. The internal annular diameter of the auto-xenograft valves (n = 3) was 19 to 23 mm (mean, 21 mm). Seeding density was between 1.0 x 10-5 and 1.2 x 10-5 cells•cm-2 (mean, 1.1 x 10-5 cells•cm-2) valve surface. Cell viability was 95.4% to 97.7% (mean, 96.8%).
Macroscopic examination of the auto-xenografts explanted after 3 months in the sheep showed on both sides of the anastomoses a fibrous overgrowth without encapsulation of the cusps. There was no vegetation, hematomas, or thrombotic material on the auto-xenografts. The leaflets showed no sign of tearing, perforation, deformation, retraction, hardness, or fibrous tissue ingrowth. (Figure 2). No massive calcification was seen on the x-ray of the explanted auto-xenografts (Figure 3). Only slight calcification could be detected at the inflow and outflow suture lines. On the outflow wall of one valve, a small local focal point of calcification was seen. No calcification was seen on any parts of the cusps. The calcium contents of all the valves were similar. The mean calcium contents of the commissural area, the middle part, and the free edge of the leaflets were 2.43 (range, 1.18 to 3.25), 2.20 (range, 1.18 to 3.65), and 2.49 (range, 1.26 to 3.70) µg•mg-1 of dry cuspal weight, respectively.
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Figure 2. A representative sample of an auto-xenograft explanted after 3 months in a juvenile sheep. There was no retraction, perforation, tearing, hardness, or deformation of the leaflets.
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Figure 3. X-ray (A) lateral and (B) frontal views of an explanted auto-xenograft, which show no calcification of the leaflets.
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Light microscopy showed no calcification on the cusps of the explanted auto-xenografts. The inflow part of the valve showed only minimal calcification at the suture line, while on the outflow wall a small area of the internal part of the media was calcified. A thin fibrous tissue covered the inflow and outflow parts of the anastomoses between the auto-xenograft and the native PA. There was no fibrous tissue overgrowth on the cusps. On the outside of the auto-xenograft, a normal inflammatory reaction was seen, while on the luminal side was a monolayer of endothelial-like cells (Figure 4), which extended to the middle of the cusps. Beyond that, only islands of endothelial-like cells were seen, while no endothelial-like cells were seen at the free edge. Von Kossa staining did not show calcification in any leaflets. Scanning electron microscopy, used to observe the endothelial cells on the luminal side of the auto-xenograft (Figure 5), showed a monolayer of endothelial cells on the inflow and outflow walls as well as at the commissural part of the cusps. The middle part showed only islands of endothelial cells, while no endothelial cells were seen at the free edge. Using factor VIII staining, it was possible to identify the cells on the inner surface of the tissue-engineered heart valve as endothelial cells (Figure 6).
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Figure 4. An autologous endothelial-like cell layer is seen on the luminal side of an explanted auto-xenograft. Fibroblasts were growing into the acellular matrix (hematoxylin and eosin stain, original magnification x40).
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Figure 5. Scanning electron microscopic appearance of a representative part of an explanted auto-xenograft with an autologous endothelial cell layer seen on the luminal side.
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Figure 6. With factor VIII staining, the cells on the inner surface of the valve were identified as endothelial cells (original magnification x40).
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In the clinical study, the mean age of the patients (n = 6) was 43.8 ± 9.2 years (range, 27 to 55 years). The internal diameter of all allografts used was 27 mm. Seeding density was 9.5 x 10-6 cells•cm-2. At discharge, the mean pressure gradient at the aortic valve was 4.7 mm Hg (range, 1.8 to 7.2 mm Hg) and at 3 months 6.2 mm Hg (range, 1.6 to 13.5 mm Hg) without evidence of valve regurgi-tation. The mean pressure gradient at the tissue-engineered heart valve was 2.2 mm Hg (range, 1.3 to 3.8 mm Hg) at discharge and 1.5 mm Hg (range, 1.1 to 2.9 mm Hg) 3 months later with trivial regurgitation in one patient. The left ventricular ejection fraction at discharge was 58.0 ± 13.6% and at 3 months 65.2 ± 9.5%. Magnetic resonance imaging showed pliable cusps with smooth movements (Figure 7).
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Figure 7. Magnetic resonance imaging of the tissue-engineered heart valve with the leaflet in a closed position. The leaflets show normal morphology.
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DISCUSSION
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Stented and stentless porcine bioprostheses are stabilized and sterilized with glutaraldehyde, which produces a significant amount of toxicity. The lack of growth of host endothelial cells on the surface of the xenograft is due to glycerol binding to residual aldehyde groups,7 while free aldehyde groups cause calcification by binding calcium.8,9 Antimineralization solutions have been investigated for controlling the free aldehyde groups. Aminooleic acid has been evaluated in stented and stentless valve prostheses with only moderate success in controlling calcification.10,11
Allografts show the most favorable clinical results, but they are not living tissue. Before implantation in patients, only 27% of the endothelial cells were found to be alive after 6 hours of warm ischemia.12 The long warm ischemia and mechanical manipulation during implantation add to a further loss of living endothelial cells.
With tissue engineering, it is now possible to replace a diseased heart valve with a living valve prosthesis. The tissue-engineered auto-xenograft does not require glutaraldehyde treatment as it is a living structure. The use of a decellularized matrix of a porcine pulmonary valve is preferred because synthetic scaffolds are not only expensive and potentially immunogenic, they also suffer from toxic degradation and inflammatory reaction.13 Recently, nonseeded allogenic and xenogenic matrices have been implanted in animals.14,15 These matrices are expected to be covered with host cells, as observed in experimental animals. Experience from clinical explants of xenografts or vascular prostheses, however, indicates that moderate to no endothelial cell growth in humans may be expected. At best, a so-called pseudointima can be seen, which is far from being a functional endothelial cell layer. This and the potential thrombogenicity of the naked collagen structures make these substitutes questionable and far from the aims of tissue engineering.
The goal of seeding the xenograft matrix with living autologous endothelial cells is to create a natural antithrombotic surface by means of a viable and functional endothelium. As an autologous surface covers the luminal side of the valve, fibrous sheathing of the cusp could be avoided. Sheathing eventually will lead to retraction or complete immobilization of the cusp. It also might induce thrombogenicity in the valves as it is believed that sheathing originates from fibrin deposition and thrombus organization. This was the first reason for not implanting an acellular matrix in animals as the outgrowth of endothelial cells is higher in animal models than in human. The second reason for coating the acellular matrix with endothelial cells was to reduce immunologic reactions, which could be an important factor in increasing calcification and degeneration of the cryopreserved allograft valves. In all the animals, a dynamic process at the level of the endothelium was observed as it is only possible to stain mature endothelial cells with factor VIII. A confluent layer of endothelial cells was seen on the luminal side of the valve, and the unstained cells could be identified by morphological criteria as immature endothelial cells.
The juvenile sheep, which is an established model, is widely used to assess the durability and functionality of new biological valve prostheses.16,17 In this study, this animal was used to implant the tissue-engineered auto-xenograft. Because of extensive experience with this model, we had no operative complications and no operative mortality. Promising results were obtained with the use of a living autologous endothelial cell layer on the inner surface of a decellularized porcine heart valve. No thrombosis on the valve surfaces and excellent leaflet function were seen in all the valves. However, long-term study of the behavior of these valves in animals is needed. Further studies are underway to determine if the porcine matrix carries infectious risks for patients through the transmission of porcine endogenous retrovirus. This potential risk was the reason we implanted auto-allografts clinically in this initial safety and feasibility study. The clinical course of all the patients was smooth and uneventful, and immunologic activity against the allogenic tissue was not observed.
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Acknowledgments
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We thank Mrs. Nickel for her excellent work with endothelial cell cultures.
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ABSTRACT
INTRODUCTION
