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Eur J Cardiothorac Surg 2003;24:358-363
© 2003 Elsevier Science NL
a Department of Cardiothoracic and Vascular Surgery, Medical School Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
b Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Medical School Hannover, Podbielskistrasse 380, D-30659 Hannover, Germany
Received 12 September 2002; received in revised form 24 April 2003; accepted 12 May 2003.
* Corresponding author. Tel.: +49-511-790-6277; fax: +49-511-790-6266
e-mail: walles{at}thg.mh-hannover.de
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Xenotransplantation • Porcine endogenous retrovirus • Tissue engineering • Bioartificial vessel grafts • Cardiovascular
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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In tissue engineering, acellular porcine scaffolds are implanted as matrices for tissue regeneration. The acellularisation process for biological matrix scaffolds might not remove all native cells or cell debris [8]. Moreover, we recently showed that after acellularisation of porcine tissue up to 2% of native DNA is still detectable within the matrix [9]. These findings might indicate an existing risk for cross-species PERV transmission after implantation of acellularised porcine tissue. However, there are no data available indicating whether these cell remnants or DNA fragments are capable for PERV transmission.
To elucidate this problem tissue engineered pulmonary arteries (PA) based on acellularised porcine pulmonary artery scaffolds were implanted into sheep for 6 months after which the graft tissue was assessed for PERV by means of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR). Furthermore blood samples were drawn regularly up to 6 months to detect PERV DNA/RNA.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.2. Scaffold acellularisation procedure
Porcine pulmonary arteries were flushed with HBSS and incubated for 48 h in 0.1% trypsin–PBS--solution (37 °C). The trypsin was changed after 24 h. Residual cells were removed flushing the scaffolds with PBS at 4 °C. For sterilization, the acellularised scaffolds were radiated with 100 Gy 
-radiation (30 min). Semithin 6 µm scaffold sections were stained with hematoxylin before implantation to control decellularization.
2.3. Cellular repopulation
Porcine acellular matrices were repopulated with ovine myo-fibroblasts (MFb) (1.2x105 cells/cm2) and EC in a bioreactor under dynamic conditions with the bioreactor rotating with 0.5 U/min for 6 h, followed by continuous perfusion with DMEM (30 ml/min) for 24 h. Three repopulation cycles were performed to each cell type. Cultivation lasted 12 days.
2.4. Immunohistology
To characterize the repopulated matrix scaffold species-specific monoclonal antibodies were used: EC by Factor VIII-related antigen (clone F8/6) and CD-31 integrin (clone JC/70A); MFb by vimentin (clone B3/7) and desmin (clone 4A/6) double-staining. Positive control consisted of untreated porcine pulmonary artery sections. Negative control was performed with mouse serum.
2.5. Infection of primary ovine EC
Isolated ovine EC were incubated with PK15 supernatants. The separations were obtained as described in detail by Patience and coworkers [5].
2.6. DNA and RNA isolation from tissue
Porcine acelluarized and explanted in vitro repopulated PA were homogenized in a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% Sarkosyl, and 0.1 M 2-ME. One milliliter homogenate was mixed with 0.1 ml 2 M sodium acetate (pH 4). One ml water-saturated phenol was added after several inversions, thoroughly mixed and 0.2 ml of 49:1 chloroform/isoamyl alcohol was added and incubated for 15 min at 4 °C. After centrifugation for 20 min at 10 000g, 4 °C the aqueous RNA containing phase was transferred into a second tube. The interphase and lower organic phase were used for precipitate DNA.
2.7. Isolation of DNA and RNA from cell cultures
Cellular DNA and RNA of cultured porcine cells (2–5x106) were prepared using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Potentially contaminating DNA in the RNA preparation was digested by DNAse treatment (for 10 µg RNA: 10 U RNAse-free DNAseI, 40 U/µl RNAse inhibitor, 1 M MgCl2, 0.1 M DTT; 15 min incubation (37 °C); phenol/chloroform extraction).
2.8. DNA precipitation
A solution of 0.3 ml of 100% ethanol was added per 1 ml solution and incubated (5 min, RT) and centrifugated (4 °C). Protein containing supernatant was removed. The DNA pellet was washed twice in 0.1 M sodium citrate and resuspended in 75% ethanol and incubated for 20 min at RT. The dried DNA pellet was dissolved in 8 mM NaOH and centrifuged at 12 000g for 10 min. The supernatant was transferred to a new tube, DNA was quantified by reading the A260 and 0.5–1 µg was added to the PCR mix.
2.9. RNA precipitation
RNA was precipitated by adding 1 ml 100% isopropanol to the aqueous phase, incubated for 30 min at -20 °C. Centrifugation at 10 000g for 10 min at 4 °C. RNA pellet was dissolved in 0.3 ml and precipitated by adding 0.3 ml 100% isopropanol. The resulting mixture was incubated for 30 min at -20 °C and centrifuged. The pellet was washed with 75% ethanol and centrifugated. The supernatant was discarded and the pellet dried. RNA was dissolved in 100–200 µl DEPC-treated water, incubated for 15 min at 55 °C and stored at -70 °C. RNA was quantified by reading the A260 and A280; 1 µg was used for RT-PCR.
2.10. PERV-specific PCR and RT-PCR
Methods were performed according to the PERV protease-specific PCR/RT-PCR described by Patience and coworkers [5]. For c-DNA synthesis 1 µg total RNA and AMV-RT were used. PCR was done with 1 µg genomic DNA. Sensitivity of our system was 1x105 cells. PK15 DNA/RNA was used as positive control. EC DNA/RNA and porcine ß-globin specific primer served as negative controls.
2.11. RT-assay to detect viral RT activity in ovine EC culture supernatant
Five milliliters of 0.45 µm filtered cell culture-supernatant was pelleted at 50 000 rpm, (30 min, 4 °C) and resuspended in 20 µl 1% NP40. 5 µl of this solution was tested in the RT-assay with BMV-RNA and BMV-specific primer. Denaturation for 5 min at 95 °C followed c-DNA synthesis. The BMV-RNA was digested with 100 µg/µl RNAse at 37 °C for 15 min. In a new tube PCR reaction described by Silver et al. [16] occurred. PCR products were separated on a 2% agarose-gel in a Ethidiumbromid-TAE-buffer.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.3. In vitro PERV-transfection studies
PERV-specific PCR in the ovine EC cell culture showed an integration of PERV sequences in the ovine genome of primary vascular EC (Fig. 2
). The RT-PCR documents a transcription of PERV sequences in the infected ovine EC. The positive RT-assay demonstrates a productive PERV infection of ovine EC (Fig. 2). Thus, ovine EC are infected by PERV and produce PERV virus.
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Histologic examinations showed intact tissue engineered PA with a confluent endothelial layer over the full graft length (Fig. 1d). Composition of vessel wall and its extracellular matrix were similar to native ovine pulmonary arteries. No signs of early calcification could be detected.
PERV-specific PCR and RT-PCR experiments in the isolated DNA and RNA of the ovine EC did not show PERV-specific sequences (Fig. 3 ). Internal controls consisting of porcine DNA and RNA showed the expected PERV-specific amplification products, thus ruling out methodological errors.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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The safety of porcine tissue for xenotransplantation has been questioned recently by Patience and Wilson who demonstrated that PERV is capable of infecting human cell lines in vitro [6]. PERV is found ubiquitous in all porcine tissues, but does not cause any disease in pigs [5]. Its infectious potential is unknown in other species including man. The risk of trans-species retroviral infections should not be underestimated, since HIV as another harmless animal retrovirus may cause severe disease in man. However, cross-species transmission of PERV to patients exposed to living pig tissue for a limited time could not be demonstrated [10,11]. Moza and coworkers showed that the glutaraldehyde treated porcine heart valve does 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 [12]. Whether chemical acellularisation of porcine tissue used for tissue engineering can transfect PERV has not been delineated so far. In this context, however, Zeltinger and coworkers observed residual cell remnants after chemical acellularisation of porcine heart valves [8]. Furthermore we demonstrated that after chemical acellularisation of porcine tissue up to 3% of native DNA is still detectable within the matrix [9]. The question, if these transferred retroviruses might trigger disease in the organ recipient is not sufficiently answered so far. The possible clinical impact of these findings justified in vivo animal studies.
We developed a chronic in vivo sheep model to simulate chimeric tissue implantation over a period of 6 months. An acellular porcine matrix was repopulated with autologous cells in vitro within 3 weeks and implanted in vivo for 6 months. Light microscopy and immunohistochemical staining were applied to control the repopulation process. PCR and RT-PCR were performed to detect PERV-specific sequences. To validate our in vivo experimental data we also established a chimeric ovine in vitro repopulation model by reseeding ovine EC on the very same acellular porcine matrix.
In this study we have shown for the first time that a primary ovine cell can be infected by PERV and converted to be an active producer of PERV particles. To our knowledge this has not been proved before by others. Our chronic in vivo model in sheep applied an acellular pECM as scaffold for in vivo reseeding with ovine EC. We found no evidence of PERV transfection neither in the chimeric cardiovascular implants following reseeding nor in the peripheral blood of experimental animals.
In our study, all isolated and cultured primary porcine vascular cells contained PERV DNA. The sensitivity of the RT-PCR assay was one infected cell in a total of 105 cells. This is in agreement with other studies in porcine cell lines showing different viral production in various cell types [13].
Chimeric cardiovascular implants composed of acellular pECM and ovine EC carry no PERV infection risk. The chosen experimental duration of 6 months is according to the work of Paradis and coworkers [14] a time period which allows even very slight PERV infections to develop and become detected. Following a retroviral PERV infection the viral titer rises within 3 weeks to levels causing virus spreading and general infection [15].
In conclusion, our developed animal model and the applied methods are suitable instruments to investigate the potential risk of PERV infection in the preclinical and clinical setting. We did not detect PERV transmission or infection in sheep after implantation of tissue engineered heart PA based on acellularised porcine matrix scaffolds repopulated with autologous cells. Thus, the chemical acellularisation process with trypsin/EDTA is sufficient enough to prevent cross-species transmission of PERV. Therefore, this matrix can be used as a scaffold for cardiovascular tissue engineering with no increased risk for cross-species transmission of PERV compared to the clinically established biological xenogenic implants.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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