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J Thorac Cardiovasc Surg 2003;126:1000-1004
© 2003 The American Association for Thoracic Surgery
Surgery for acquired cardiovascular disease |
a Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany
b LEBAO (Leibniz Research Laboratories for Biotechnology and Artificial Organs), Hannover, Germany
Received for publication August 1, 2002; revisions received August 28, 2002; accepted for publication December 30, 2002.
* Address for reprints: Rainer G. Leyh, MD, PhD, Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl Neuberg St. 1, 30623 Hannover, Germany
leyh{at}thg.mh-hannover.de
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Abstract |
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METHODS: Acellularized porcine pulmonary valve conduits (n = 3) and in vitro autologous repopulated porcine pulmonary valve conduits (n = 5) were implanted into sheep in the pulmonary valve position. Surgery was carried out with cardiopulmonary bypass support. The animals were killed 6 months after the operation. Blood samples were collected regularly up to 6 months after the operation and tested for porcine endogenous retrovirus by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. In addition, explanted tissue-engineered heart valves were tested for porcine endogenous retrovirus after 6 month in vivo.
RESULTS: Porcine endogenous retrovirus DNA was detectable in acellularized porcine heart valve tissue. However, 6 months after implantation of in vitro and in vivo repopulated acellularized porcine heart valve scaffolds, no porcine endogenous retrovirus sequences were detectable in heart valve tissue and peripheral blood.
CONCLUSION: Acellularized porcine matrix scaffolds used for creation of tissue-engineered heart valves do not transmit porcine endogenous retrovirus.
The acellularization process for biological matrix scaffolds might not remove all native cells or cell debris.8 Moreover, we recently showed that after acellularization of porcine tissue, up to 2% of native DNA is still detectable within the matrix.9 These findings might indicate an increase risk for cross-species PERV transmission after implantation of acellularized porcine tissue. However, there are no data available indicating whether these cell remnants or DNA fragments are capable of PERV transmission.
To elucidate this problem, tissue-engineered heart valves based on acellularized porcine heart valve scaffolds were implanted into sheep for 6 months, after which the valvular tissue was assessed for PERV by means of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR). Furthermore, blood samples were drawn regularly for up to 6 months to detect PERV DNA/RNA.
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Methods |
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Cell isolation and culture
The technique of cell isolation and cell culture have been described in detail elsewhere.1,10 For the generation of autologous cell culture, short segments of the right carotid artery were harvested from 5 lambs.
Cell seeding
The acellularized porcine valve conduits (n = 5) were seeded first with myofibroblasts and coated with endothelial cells, resulting in a uniform cellular restitution of the pulmonary valve conduit surface. Three separate cycles of myofibroblast and endothelial cell seeding were performed. In each cycle 1 x 106 myofibroblasts or endothelial cells were seeded onto the xenogeneic pulmonary valve scaffold fixed into a bioreactor and cultured in static nutrient medium (Dulbecco's modified Eagle's medium, GIBCO, Karlsruhe, Germany) for 4 hours (37°C, 5% CO2), followed by rotating of the bioreactor (12 hours, 0.1 rpm). Samples of the conduit were taken to document seeding of myofibroblast and endothelial cells.
Pulmonary valve conduit replacement
Eighteen days after the initial cell seeding, the repopulated pulmonary valve conduits were implanted into the same 5 lambs from which the initial vessels harvested (age 10 to 12 weeks, weight 25 to 30 kg), and the acellularized porcine heart valve scaffolds were implanted in the remaining 3 sheep. The technique has been described in detail elsewhere1; in brief, the heart was exposed by a left anterolateral thoracotomy, by means of femoral arterial and right atrial venous cannulation, and normothermic cardiopulmonary bypass was established. With the heart beating, the pulmonary artery was transected, all 3 native leaflets removed, and the valve conduits implanted. All animals received humane care in compliance with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (National Institutes of Health publication No. 85-23, revised 1985).
Histology and immunohistochemistry
The acellularization treatment was documented by hematoxylin-eosin staining. Histological characterization was performed by means of standard hematoxylin-eosin and Movat-Pentachrom staining. Immunohistochemical staining for endothelial cell characterization was performed by use of the avidin-biotin-peroxidase technique (factor VIII-related antigen; primary antibody, von Willebrandt factor; clone 8/86, DAKO). For characterization of myocytes and fibroblasts, a double immunofluorescence technique with monoclonal antibodies against myocytes (monoclonal desmin antibody; clone C-18, Santa Cruz Biotechnology, Santa Cruz, Calif) and fibroblasts (polyclonal vimentin antibody; clone Vim H-84, Santa Cruz Biotechnology) were used.
DNA and RNA isolation from tissue
Porcine acellularized and explanted in vitro and in vivo repopulated heart valves were homogenized in a solution containing 4 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, 0.5% Sarkosyl, and 0.1 mol/L 2-mercaptoethanol. Homogenate (1 mL) was mixed with 0.1 mL 2 mol/L sodium acetate (pH 4). Water-saturated phenol (1 mL) was added after several inversions and thoroughly mixed, and 0.2 mL of 49:1 chloroform/isoamyl alcohol was added. Incubation occurred for 15 minutes at 4°C. After centrifugation for 20 minutes at 10,000 g, 4°C, the aqueous RNA-containing phase was transferred in a second tube. The interphase and lower organic phase were used for precipitate DNA.
DNA precipitation
Pure (100%) ethanol (0.3 mL) was added per 1 mL of solution and incubated for 5 minutes at room temperature and centrifuged at 2000 g for 5 minutes at 4°C. Protein containing supernatant was removed. The DNA pellet was washed twice in 0.1 mol/L sodium citrate and resuspended in 75% ethanol, then incubated for 20 minutes at room temperature. The dried DNA pellet was dissolved in 8 mmol/L NaOH and centrifuged at 12,000 g for 10 minutes. The supernatant was transferred to a new tube, DNA was quantified by reading the A260, and 0.5 to 1 µg was added to the PCR mix.
RNA precipitation
RNA was precipitated by adding 1 mL of 100% isopropanol to the aqueous phase, incubated for 30 minutes at -20°C, then centrifuged 10,000 g for 10 minutes at 4°C. RNA pellet was dissolved and precipitated by adding 0.3 mL 100% isopropanol. Incubation was for 30 minutes at -20°C. The pellet was washed with 75% ethanol. The supernatant was discarded and the pellet dried. RNA was dissolved in 100 to 200 µL diethylpyrocarbonate-treated water, incubated for 15 minutes at 55°C, and stored at -70°C. RNA was quantified by reading the A260 and A280; 1 µg was used for RT-PCR.
In vitro isolation of peripheral blood monocytes from whole blood
Ficoll-Paque gradient was prepared according to the manufacturer's instructions. Anticoagulant-treated blood was layered on the Ficoll-Paque solution and centrifuged at 400 g for 40 minutes at 20°C. Differential migration during centrifugation results in the formation of layers containing different blood cell types. First, the upper layer of plasma was discarded. Then the lymphocyte layer was transferred to a clean centrifuge tube. Two washing steps followed, adding 3 volumes of balanced salt solution; centrifugation at 100 g for 10 minutes at 20°C followed that. The supernatant was removed, and the lymphocytes in pellets were homogenized in denaturing solution.
Isolation of DNA and total RNA of peripheral blood monocytes and tissue
The cusps of 1 heart valve and Ficoll-Paque–isolated peripheral blood monocytes (PBMCs) were homogenized in a denaturing solution containing 4 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, 0.5% N-lauroyl-sarcosine (Sarkosyl), and 0.1 mol/L 2-mercaptoethanol. One milliliter of homogenate was mixed with 0.1 mL of 2 mol/L sodium acetate (pH 4). After several inversions, 1 mL of water-saturated phenol was added and thoroughly mixed, and 0.2 mL of 49:1 chloroform/isoamyl alcohol was added. The suspension was incubated for 15 minutes at 4°C. After centrifugation at 10,000 g for 20 minutes at 4°C, the aqueous RNA-containing phase was transferred into a second tube. The interphase and lower organic phase were used to precipitate DNA.
Purification of viral RNA from plasma
For purification of viral RNA from human plasma, Qiagen QiAamp viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany) was used. Viral RNA preparation was done according to the manufacturer's instructions.
PERV-specific PCR and RT-PCR
These methods were performed according to the PERV pol-specific PCR/RT-PCR of Patience and colleagues5 and porcine-specific RT-PCR of Heneine and associates.11 For cDNA synthesis, 1 µg of total RNA and avian myeloblastosis virus–reverse transcriptase (Boehringer Mannheim, Germany) was used. PCR was done with 1 µg of DNA. PCR products were separated on 2% agarose gel in an ethidium bromide–tris-acetate-EDTA buffer.
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Results |
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Histological and immunohistological evaluation
The acellularization procedure resulted in a complete cell. Tissue-engineered valves showed a complete seeding of myofibroblasts and endothelial cells of the valve (seeding controls not transplanted) and conduit wall, evaluated by positive staining for von Willebrandt factor and vimentin desmin double immunofluorescence technique. Furthermore, the interstitium of the tissue-engineered pulmonary valve conduit showed a fibroblast and myocyte population similar to native ovine valve tissue. The Movat pentachrome stain showed amounts of stainable collagen, glycosaminoglycans, proteoglycans, and elastin similar to native ovine valve tissue.
PERV sequences in heart valve tissue
Two cusps of 1 heart valve and half of the surrounding annulus from 3 different porcine heart valves were used to isolate RNA and DNA. In acellularized porcine scaffolds PERV DNA was detectable. However, 6 months after implantation no PERV-specific sequences (PERV-specific PCR and RT-PCR) could be amplified in in vivo and in vitro repopulated heart valves (Figure 1).
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Discussion |
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The safety of porcine tissue for xenotransplantation has been questioned recently by Patience and colleagues5 and Wilson and coworkers,6 who demonstrated that PERV is capable of infecting human cell lines in vitro. However, cross-species transmission of PERV to patients exposed to living pig tissue for a limited time could not be demonstrated.13,20 Moza and coworkers21 showed that glutaraldehyde-treated porcine heart valves do not carry PERV DNA, and patients receiving porcine heart valves do not show any signs of PERV infection up to 3 years after implantation of glutaraldehyde treated porcine heart valves. Whether chemical acellularization of porcine tissue used for tissue engineering prevents PERV infection has not been delineated so far. In this context, however, Zeltinger and coworkers8 observed residual cell remnants after chemical acellularization of porcine heart valves. Furthermore, we demonstrated that after chemical acellularization of porcine tissue, up to 2% of native DNA is still detectable within the matrix.9 The possible clinical impact of this finding justified in vivo animal studies. According to our PCR and RT-PCR results, no fragments of PERV sequences were detected in tissue-engineered heart valves after 6 months of implantation. Furthermore, we did not detect PERV RNA-DNA isolated from lymphocytes and plasma of sheep that underwent heart valve replacement with tissue-engineered heart valves based on acellularized porcine tissue. These results provide evidence that acellularized porcine heart valve scaffolds used for heart valve tissue engineering do not transmit PERV to sheep.
Limitation of the study
Cross-species transmission of PERV has not been demonstrated in vivo. Although PCR and RT-PCR are sensitive tools for detecting PERV sequences, it could be argued that the failure to detect cross-species transmission of PERV in this study is a methodological problem. However, the implantation time of 6 months should be sufficient to detect any PERV infection with the methods used in this study. Other methods like electron microscopy, giving the capability to proof PERV transmission by direct proof of PERV virus, would have added further information.
In conclusion, we did not detect PERV transmission or infection in sheep after implantation of tissue-engineered heart valves based on acellularized porcine matrix scaffolds. The preliminary data from this study indicate that the chemical acellularization process with trypsin/EDTA is sufficient enough to prevent cross-species transmission of PERV in a sheep model. However, further studies are mandatory to draw the definite conclusion that the porcine matrix can be used as a scaffold for heart valve tissue engineering with no increased risk of cross-species transmission of PERV.
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