Asian Cardiovasc Thorac Ann 2003;11:153-156
© 2003 Asia Publishing EXchange Ltd
Tissue-Engineered Heart Valve on Acellular Aortic Valve Scaffold: In-Vivo Study
Dong-e Zhao, MD,
Ruo-bing Li, MD,
Wei-yong Liu, MD,
Gang Wang, MD,
Shi-qiang Yu, MD,
Cheng-wei Zhang, MD,
Wen-sheng Chen, MD,
Geng-xu Zhou, MD
Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, Xian, People’s Republic of China
For reprint information contact: Dong-e Zhao, MD Tel: 86-10-68212211-75558 email: zhaodonge{at}hotmail.com Department of Cardiovascular Surgery, General Hospital of Armed Police Forces, Beijing 100039, People’s Republic of China.
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ABSTRACT
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The feasibility of constructing a tissue-engineered heart valve on an acellular porcine aortic valve leaflet was evaluated. A detergent and enzymatic extraction process was developed to remove the cellular components from porcine aortic valves. The acellular valve leaflets were seeded for 7 days in vitro with cells from canine arterial wall and endothelial cells. The constructs were implanted into the lumens of 6 canine abdominal aortas to assess the reconstruction of the valve leaflets. It was found that all cellular components had been removed from the porcine aortic valves. The valve leaflets were completely reconstructed at the end of the 10th week in vivo. Scanning electron microscopy showed that the valve leaflets were partially covered with endothelial cells. It was concluded that porcine aortic valves can be decellularized by the detergent and enzymatic extraction process and it is feasible to construct a tissue-engineered heart valve in vivo on an acellular valve scaffold.
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INTRODUCTION
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The most common surgical therapy for heart valve disease consists of valve replacement, with more than 60,000 implantations annually in the United States and 170,000 worldwide.1 Valve replacement surgery is effective and has been shown to significantly alter the course of valvular disease, but it has some associated shortcomings. Mechanical valves need lifelong anticoagulation and they are susceptible to infection. Bioprosthetic valves do not require anticoagulation but they have limited durability and are subject to progressive tissue deterioration. None of the available valve replacements has the capacity for growth, which is necessary in pediatric patients. Thus, efforts have been made to use the techniques of tissue engineering to create an ideal valve substitute. There have been two approaches to tissue engineering of heart valves over the last 7 years. The first involves decellularized heart valves with autologous cells seeded onto them.2,3 The second approach is to seed cells on synthetic biodegradable scaffolds in the shape of the valve leaflets.4,5 We describe a method for enzymatic decellularization of xenogenic porcine aortic valves and their subsequent seeding with autologous myofibroblasts and endothelial cells. The constructs were implanted into the lumens of canine abdominal aortas to assess the reconstruction.
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MATERIALS AND METHODS
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Twenty porcine aortic valve leaflets (14 for decellularization and 6 for calcium content determination) were obtained from a local slaughterhouse. The leaflets were excised and freed from adherent fat and aortic wall. They were stored immediately in Hank’s balanced salt solution at 4°C; warm ischemic time was less than 10 min. After transfer to the laboratory, 14 leaflets were placed in a solution of 1% tert-octylphenyl-polyoxyethylen (Triton X-100, Sigma, USA) with hypotonic Tris chloride buffered solution (Tris-Cl 0.001 mol•L-1, HCl 0.077 mol•L-1, Triton X-100 100 mL•L-1; pH 8.0) at 4°C. After 12 hours, the valves were placed in a hypertonic solution (KCl 1.5 mol•L-1, Triton X-100 100 mL•L-1) for 12 hours, and in 1% Triton X-100 for another 12 hours at 4°C. Then the valves were treated with DNAase I (0.025 mg•mL-1; Sigma, USA) and RNAase A (0.01 mg•mL-1; Sigma, USA) in a Tris chloride buffered solution with 0.02% EDTA (ethylenediaminetetraacetic acid) for 12 hours at 37°C. All steps were conducted under continuous shaking. The valves were washed with phosphate-balanced solution several times, sterilized with Co60 gamma-irradiation, and stored in Hank’s balanced salt solution at 4°C prior to further testing and seeding. Specimens were prepared for scanning electron microscopy and light microscopy.
To obtain autologous cells, a 3-cm segment of the right carotid artery was harvested from 6 60–70-day-old dogs weighing 12–16 kg. The segments were filled with 0.1% collagenase I (Sigma, USA) in Dulbecco minimum essential medium (DMEM; HyClon, USA) at 37°C. After 15 min, the arteries were flushed with 20 mL DMEM. The endothelial cells were pelleted by centrifugation for 6 min at 300g, resuspended in 5 mL DMEM with 10% fetal bovine serum (HyClone, USA), 100 µg•mL-1 penicillin, 5 ng•mL-1 endothelial growth factor (Sigma, USA), and 5000 µg•mL-1 heparin (Sigma, USA), and placed in culture flasks at a density of 10,000 cells/cm2. The medium was changed every 3 days. The cells were cultured under 5% CO2 in an incubator maintained at 37°C for 3–4 weeks. The rest of the artery was minced, and 2-mm pieces of arterial wall were placed in Petri dishes in DMEM for 9 days. Myofibroblasts grew on the surface of the culture dish and were subcultivated in culture flasks. After cell expansion, myofibroblasts and endothelial cells were seeded onto the acellular porcine aortic valves in a sequential seeding process that involved 4 days of static seeding on the upper surface of valves with myofibroblasts at a density of 300,000 cells/cm2. Then 3 days of endothelial cell seeding at a density of 300,000 cells/cm2 was performed sequentially.
The tissue-engineered porcine aortic valve leaflets were implanted into the lumens of the abdominal aortas of the 6 dogs that had undergone previous harvest of the right carotid artery. Anesthesia was induced with ketamine and maintained by infusion of propofol. Retroperitoneal access to the infrarenal abdominal aorta was achieved. The aorta was snared over a 2-cm segment distal to the bifurcation of the renal artery, and it was partially incised transversely. The valve leaflets were sutured into the lumen of the aorta with 5/0 polypropylene suture (Ethicon; J & J Medical, Shanghai, China). Four dogs received one piece of valve leaflet, and 2 were given an extra piece of leaflet for the determination of calcium. The aorta was released and the incision was closed. All animals survived the operative procedure and received humane care in the laboratory animal center.
One dog was sacrificed at the end of the 4th, 6th, 8th, and 10th week; the 2 animals with 2 leaflets were also sacrificed the end of the 10th week for detection of calcium. No thrombus was found on gross inspection. The specimens were fixed with 4% paraformaldehyde. Standard hematoxylin-eosin staining was used and the sections were analyzed by light microscopy. Immunohistochemistry was performed using the avidin-biotin-peroxidase technique. Endothelial cells were detected by the presence of factor-VIII-related antigen (von Willebrand factor; Boxter, China) and the myofibroblasts were characterized by the presence of smooth-muscle actin (Boxter, China). The calcium content of 5 of the explanted valve leaflets (1 routine specimen and the 4 extra specimens) was measured by atomic absorption spectrometry and compared with the levels of calcium found in 6 fresh porcine aortic valves and 6 acellular valves.6
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RESULTS
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The decellularization procedure resulted in complete cell loss in the valves (Figures 1a
and 1b). Tissue-engineered valves showed confluent seeding of myofibroblasts and endothelial cells (Figure 2a), which was confirmed by positive staining for von Willebrand factor and smooth-muscle actin (Figure 2b).
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Figure 1. (a) Acellular valve histology (hematoxylin-eosin stain, original magnification x200). (b) Transmission electron microscopy of the acellular porcine aortic valve, showing no cellular component in the valve matrix (original magnification x2,500).
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Figure 2. Cell seeding on the acellular valve. (a) Myofibroblasts seeded on the valve (hematoxylin-eosin stain, original magnification x200). (b) Endothelial cells seeded on the valve, showing positive immunohistochemical staining for von Willebrand factor (peroxidase stain, original magnification x400).
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The tissue-engineered heart valves were completely covered by seeded cells. At the end of the 4th week, some myofibroblasts grew into the matrix of the tissue-engineered valve (Figure 3). At the end of the 10th week, the tissue-engineered valve leaflets were almost completely remolded by the seeded cells (Figure 4a). Scanning electron microscopy and immunohistochemistry staining for von Willebrand factor showed that the surfaces of the valves were partially covered by endothelial cells (Figures 4b and 4c). Positive expression of smooth-muscle actin in the matrix of the valves demonstrated reconstruction of the valves by myofibroblasts (Figure 4d).
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Figure 3. Histology of a tissue-engineered valve at the end of the 4th week (hematoxylin-eosin stain, original magnification x400). The valve was covered by seeded cells and some myofibroblasts grew into the matrix.
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Figure 4. Histology and immunohistochemical staining of a valve at the end of the 10th week. (a) Hematoxylin-eosin staining shows reconstruction of the valve by the seeded cells (original magnification x400). (b) Scanning electron microscopy (original magnification x700) and (c) positive immunohistochemical staining for von Willebrand factor (original magnification x400) demonstrate that the surfaces of the valve are partially covered by endothelial cells. (d) Positive expression of smooth-muscle actin can be seen in the matrix of the valve (peroxidase stain, original magnification x400).
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The mean (± standard deviation) calcium content of 5 valves explanted after 70 days was 0.89 ± 0.61 mg per gram dry weight. The calcium level found in 6 fresh porcine aortic valves was 0.61 ± 0.38 mg per gram dry weight. The 6 acellular valves had a mean calcium level of 0.74 ± 0.53 mg per gram dry weight. There was no significant difference in calcium levels between the 3 groups (t test: p > 0.05).
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DISCUSSION
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The ability to restore heart valve function with a living self-repairing replacement would have a profound impact on cardiac surgery. This goal has been partially achieved by the Ross procedure. Previous reports of tissue engineering focused on synthetic biodegradable scaffolds such as polylactic/glycolic acid and polyhydroxyalkanoate, but there are many difficulties including poor mechanical characteristics of the scaffolds, poor adherence of the seeded cells, and hyperplasia of the fibrous tissue.4,5 Steinhoff 3 and O’Brien7 have reported encouraging results with tissue-engineered heart valves on acellular valvular scaffold, but all of these valves were implanted in the pulmonary artery.
Glutaraldehyde is routinely used to preserve tissue from tearing and to overcome its antigenicity. The disadvantage is its significant toxicity. For tissue engineering, a glutaraldehyde-free decellularized porcine matrix is preferred for preparation of a cell-free collagen scaffold for better autologous cell binding. In this study, a 3-step decellularization process was established for porcine aortic valve, without using glutaraldehyde. Myofibroblasts and endothelial cells grew well on the surface of the scaffolds in vitro. Histochemical examination of the leaflets explanted after 70 days in the aortic lumen confirmed that the seeded myofibroblasts could grew into the matrix of the valves and synthesize collagen. The valve leaflets were also partially covered by endothelial cells, which is important for valve function. The seeded cells were not marked in this experiment, so it could not be established whether the cells in the leaflets after 10 weeks were new ones or those originally seeded. Elkins and colleagues2 showed that a decellularized valve could be recellularized in vivo without in-vitro cell seeding. It is still unknown whether this has any advantages over cell seeding in vitro, and further investigations should be undertaken.
No calcification occurred during 70 days of implantation. The lack of calcification emphasizes the absence of damage to the collagen matrix, which might have resulted from the decellularization and irradiation treatments, as damaged collagen sites have been implicated as the nidus for mineralizaiton. There is a tendency for the calcium content at explant to be higher than the level after decellularization and there are limited specimens at explant. We will continue to investigate the long-term (6 and 12 months) results regarding this aspect.
All of our preliminary results are encouraging and suggest that the acellular xenograft could be an appropriate scaffold for a tissue-engineered heart valve, but there are still many questions that should be addressed. Several decellularization processes for the porcine valve have been reported, but which one is best?3,7,8 As immunogenicity is a major cause of calcification, can all traces of cells and antigens be completely removed from the scaffolds while preserving the mechanical properties of the valve? The long-term outcome of these grafts remains unclear and further studies are needed. The most appropriate seed cells (venous, arterial, or stem cells) for tissue-engineered heart valves need to be determined, as well as the optimal conditions for in-vitro seeding, for example, use of a pulsatile device.8–10 We are performing further studies focusing on these problems.
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REFERENCES
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ABSTRACT
INTRODUCTION
