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Ann Thorac Surg 2007;83:1501-1507
© 2007 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
b Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
c Department of Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
d Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan
e Cardio, Inc, Kobe, Japan
Accepted for publication November 28, 2006.
* Address correspondence to Dr Ota, Division of Cardiovascular Surgery, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan (Email: toota-cvs{at}umin.ac.jp).
| Dr Uchimura discloses a financial relationship with Cardio, Inc.
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Abstract |
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Methods: Fresh porcine aortic valves were decellularized using polyethylene glycol and gamma irradiation. These were evaluated by histologic, biochemical (DNA, solubilized protein and collagen content), mechanical (strength test, transmission electron microscopy) and immunologic (porcine endogenous retrovirus and the 
-1.3 galactosyl epitope) analyses. Implantations into the subcutaneous tissue of rats (1 week, n = 10; 2 months, n = 10) and into the descending aorta of dogs (2 months, n = 6; 6 months, n = 3) were used as in vivo studies.
Results: Complete decellularization was confirmed by histologic examination and by determining the DNA and solubilized protein content. The decellularized valve showed no significant differences in its mechanical strength or collagen content compared with native porcine tissues. The ultrastructure was well preserved in transmission electron microscope images. The DNA sequence of a porcine endogenous retrovirus and the -1.3 galactosyl epitope were eliminated after the decellularizing process. No acute rejection and little calcification was noted in the rat model. In the dog model at 2 months, the surface of the graft was completely covered with a monolayer of endothelial cells. In addition, several layers of vimentin-positive cells lay under the endothelial cells. At 6 months after implantation, many smooth muscle cells, monolayer endothelial cells, and some vasculogenesis were seen.
Conclusions: The decellularizing method provided low immunogenicity, low risk of unknown infections, and was little subject to calcification. The decellularized tissues showed acceptable durability and recellularization.
Valve replacement is the most common treatment for valve diseases worldwide. Recently, bioprosthetic valves have been used extensively in cardiac surgery [1]. Although these valves are in widespread use to treat valve diseases, unresolved problems remain related to durability [2].
Almost all bioprosthetic valves used in clinical applications at present are crosslinked with glutaraldehyde. Calcification is the most frequent cause of the clinical failure of glutaraldehyde-crosslinked valves [3]. Calcification on the cusp is usually followed by stenosis or regurgitation and can result in graft failure. Furthermore, the glutaraldehyde-crosslinked valves have no viable tissues and therefore cannot regenerate or grow. In addition, the residual cytotoxicity of the aldehydes [4] and the inability of recipient cells to migrate into the fixed matrix hinder the population of these grafts with host tissue.
To overcome these limitations, tissue-engineered valves using an acellular graft have been developed. Decellularized valves have low immunogenicity, high resistance to calcification, and show recellularization by the host mesenchymal cells that could result in regeneration of the extracellular matrix (ECM); however, in vivo implantations of these valves have shown poor results so far [5].
We have developed a new method to decellularize a porcine aortic valve using polyethylene glycol (PEG) as the detergent and gamma irradiation. PEG is a biocompatible amphiphilic polymer that is nontoxic for humans and is a good detergent for decellularizing tissues [6]. Gamma irradiation is in widespread use as a method for sterilizing medical equipment [7]. This study used several biochemical analyses and preliminary in vivo tests to evaluate the biologic properties of the new decellularized tissue.
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Material and Methods |
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Mechanical Strength of Decellularized Valves
The mechanical strength of the decellularized walls was measured (n = 10) as described [8]. A tensile tester (Tensilon RTC-1150A, Orientec Co, Tokyo, Japan) was used perform uniaxial stretching on specimens of 30 mm length and 5 mm breadth at a strain rate of 10 mm/min. The maximum breaking point was determined. As a control, porcine native aortic walls were used (n = 10).
Transmission Electron Microscopy
Specimens were fixed at room temperature in 2% glutaraldehyde (Sigma, St. Louis, MO) in cacodylate buffer for 30 minutes. Specimens were further fixed in osmium tetroxide (Sigma), dehydrated in graded alcohol, and embedded in araldite. Ultrathin sections were stained with lead acetate and lead citrate (Serva, Oxon, United Kingdom). Random sampling of fields of view was used.
Biochemical Examination
To confirm decellularization of our grafts quantitatively, the amount of DNA and solubilized protein content were determined, which should reflect an amount of cell components in a tissue. The collagen content, which was the main element of ECM scaffold, was quantified to make sure that our decellularizing process did not reduce collagen in the tissue.
A 4-hydroxyproline assay was used to measure the collagen content in the decellularized valves, as described [9]. The amount of DNA was determined by the Hoechst assay (DNA quantitative kit, Hokudo, Sapporo, Japan). The solubilized protein was quantified by the Bio-Rad Protein Assay Kit I (Bio-Rad Laboratories, Hercules, CA). The levels of these components were compared with those of native porcine aortic roots. Each assay had 10 samples.
Polymerase Chain Reaction Detection of the Porcine Endogenous Retrovirus
DNA was isolated from tissues using the DNeasy Tissue Kit (QIAGEN, Valencia, CA), and 50 ng of DNA was used in polymerase chain reactions (PCRs). PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) with 10 minutes at 95°C, 15 seconds at 95°C, 15 seconds at 58°C, and 1 minute at 72°C for 40 cycles, followed by 7 minutes at 72°C. PCR reactions were performed in 20 µL volumes using rTaq polymerase (TaKaRa, Shiga, Japan). Porcine DNA was used as a positive control. Test accuracy was verified by amplifying porcine and dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the specific primers were as follows:
Rat and Dog Implantation
A rat model was used to evaluate acute rejection and calcification. Lewis strain rats (Seac Yoshitomi Ltd, Fukuoka, Japan) were anesthetized, and the decellularized graft (wall and cusp: 5 x 5 mm) was implanted subcutaneously. Ten rats were sacrificed 1 week after implantation (Rat1W group) to evaluate acute rejection, and 10 were sacrificed at 2 months (Rat2M group) to evaluate calcification. Histologic examinations were as described in the next section, and the determination of calcium in the tissues was as described previously [10]. The controls were porcine native aortic tissues for the acute rejection test (n = 10), and glutaraldehyde-treated porcine aortic tissues for the calcification test (n = 10).
The decellularized graft walls were implanted into the descending aortas of 9 female mongrel dogs (weighing 20 to 25 kg). Anesthesia and heparinization were performed as described [11]. The thoracic descending aorta was exposed through a left thoracotomy at the fourth intercostal space. The distal and proximal aorta was clamped while a temporary bypass was placed. The decellularized graft conduit was interposed with 5-0 polypropylene continuous suture. Histologic examinations were performed at 2 (Dog2M group, n = 6;) and 6 months (Dog6M group, n = 3) after implantation.
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No .85-23, revised 1985).
Quantitative Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the cellular population in the explanted tissues from the Dog2M and Dog6M groups. The expression of von Willebrand factor (vWF), vascular smooth muscle -actin 2 (ACTA2), smooth muscle 22 (SM22), and vimentin was determined by quantitative real-time RT-PCR, as described [12]. The sequences of the specific primers were as follows:
: (forward) 5'-AAGCTGGTCAACAGCCTGTATCC3', (reverse) 5'-ATAGAGGTCGACGGTCTGGAACA-3'; and
Histology and Immunohistochemistry
Cryosections (5-µm thick) of tissues were treated with hematoxylin and eosin, elastica van Gieson, or von Kossa staining. Immunohistochemical staining was performed using monoclonal antibodies for factor VIII–related antigen (DAKO, Kyoto, Japan), vimentin (DAKO), -smooth muscle actin (-SMA; clone HHF35, DAKO), and 1.3-galactosyl (1.3-Gal; Griffonia simplicifolia Lectin I-B4 Isolectin, VECTOR, Burlingame, CA).
Statistical Analysis
All values are expressed as the mean ± standard deviation. The statistical differences in all data were determined by a two-way analysis of variance. A value of p < 0.05 was considered to be statistically significant.
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Results |
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Biochemical Examination
The dry weight collagen content was 283.5 ± 23.7 µg/mg in the native porcine tissue and 237.9 ± 126.3 µg/mg in the decellularized tissue (Fig 2B). The dry weight amount of DNA in the native valve was 31.7 ± 3.5 µg/g in the wall and 41.1 ± 4.3 µg/g in the cusp, and in the decellularized valve, 1.16 ± 0.23 µg/g in the wall (p < 0.05) and 2.98 ± 1.65 µg/g in the cusp (p < 0.05; Fig 2C). The dry weight solubilized protein content was 117.0 ± 42.0 mg/g in the native valve and 0.45 ± 0.06 mg/g in the decellularized valve (p < 0.05; Fig 2D).
Immunogenicity and Infection Risk of the Decellularized Valves
Porcine endogenous retrovirus (PERV) is considered a possible infection risk from transplanting porcine tissue into humans, and 1.3-Gal is a porcine epitope that elicits acute rejection in human hosts. We hoped that our decellularizing process would eliminate these risks. Generally, PERV is detected in all porcine native tissue. To detect PERV in the decellularized valve, we transplanted the decellularized valve into a dog descending aorta model and amplified PERV sequences by PCR using DNA extracted from the preimplant decellularized graft (n = 5) and tissues excised at 2 months (n = 5) and 6 months (n = 1) from dogs as templates. PERV was not detected by PCR in the decellularized valves (Fig 3) or in the transplanted valves retrieved from dog descending aorta 2 and 6 months after implantation.
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1.3-Gal, an oligosaccharide chain responsible for the hyperacute rejection of pig tissue [13]. The porcine native tissue (Fig 4A) and porcine tissue treated with the decellularizing process, except the gamma irradiation step (Fig 4B), did have detectable 1.3-Gal. The porcine native tissue irradiated with 100 kGy gamma rays was negative for 1.3-Gal (Fig 4C). No 1.3-Gal was detected in the decellularized valve (Fig 4D). These results show that the 1.3-Gal epitope was destroyed by the gamma irradiation.
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Three dogs unexpectedly died (Dog2M group, 1; Dog6M group, 2) from infection in 1 and unknown causes in 2. Five dogs were sacrificed at 2 months and 1 at 6 months.
In the Dog2M group, a monolayer of factor-VIII positive cells was present on the surface of the grafted tissue, and several layers of infiltrating cells were vimentin-positive (Fig 6A,
6B, 6C). In the Dog6M group, more abundant cell repopulation was seen (Fig 6D). The surface of the tissue was covered with a monolayer of factor-VIII–positive cells (Fig 6G). Factor-VIII positiveness and morphologic findings supported that those cells were endothelial cells (ECs). Many -SMA positive cells were spread throughout the tissue (Fig 6F). In addition, vasculogenesis, indicating a proper remodeling, was observed in the middle of the graft (Fig 6E).
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14.2% ± 5.1% (p = 0.07). In the Dog6M group, the levels were vWF, 91.7%; vimentin, 129.4%; ACTA2, 94.3%; and SM22, 60.4%. |
Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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The decellularizing treatment with PEG and DNase alone was insufficient, however, because the porcine aortic roots had a complicated architecture that resulted in uneven tissue thickness (data not shown). We then used gamma irradiation, which could operate upon entire tissues evenly. Gamma irradiation causes cell injury through two mechanisms: one is direct cytotoxicity through the destruction of DNA [17]; the other is indirect cytotoxicity caused by reactive oxygen species, such as hydroxyl radicals, which are created by the interactions between the gamma rays and water molecules [18].
The complete decellularization in this study was accomplished by combining PEG, DNase, and gamma irradiation. The ECM of the decellularized valve was well preserved, and we observed by TEM that even the ultrastructure of the collagen and elastin was maintained. Although the collagen content of the decellularized valve was well maintained compared with native tissues, the DNA and the solubilized protein contents, which reflect the presence of cellular materials, were significantly diminished in the decellularized valve.
The concept of decellularization possibly provides some advantages in xenotransplantation, but the decellularized tissues without the glutaraldehyde fixation could be a source of infection or rejection. We performed investigations for those aspects (ie, PERV, 1.3-Gal) in porcine decellularized tissue. Generally, the dosage of gamma rays used to sterilize medical materials is 25 kGy [7]. No organism can survive the dose of 100 kGy that we used; therefore, the decellularized valves undergoing this process should be completely sterile. This is particularly important for PERV, which has been identified as a possible infection risk for human cells [19, 20].
Concern about the infection by PERV or other unknown infectious diseases is important to address in xenotransplantation therapies, particularly because xenotransplantation has recently thrived owing to the shortage of human allografts. Pigs are a particularly promising source of xenografts because their tissues can be obtained at low cost, are readily available, and have considerable compatibility with the human heart. In the present study, the DNA sequences of PERV were eliminated from the decellularized tissue as assessed by PCR, and this was confirmed by the lack of detectable PERV in the tissue that had been implanted into dogs. Therefore, our tissue-engineered xenovalves are promising in terms of their biologic safety.
The gamma irradiation also affected the oligosaccharide chains of the graft. The 1.3-Gal epitope is known to induce the hyperacute rejection of porcine xenotransplants [21, 22]. We demonstrated that the 1.3-Gal epitope was eliminated by our gamma irradiation treatment. We also expected that this treatment would destroy cell remnants, including other oligosaccharide chains, which might cause calcification [14]. Consequently, we can expect our graft would possess low immunogenicity associated with calcification, which was supported by the results of the rat experiments. Our graft will not induce hyperacute rejection related to 1.3-Gal when transplanted into humans, simply because it has no 1.3-Gal epitope.
Generally, decellularized tissues are somewhat fragile because structural components have been removed. The results of the uniaxial mechanical test in the present study show that the decellularized tissue potentially possessed mechanical strength comparable to the native tissue, which was probably because our decellularizing process preserved the scaffold structure of the ECM very well. Although a mechanical test does not reflect accurate mechanical property of a material, the graft implanted into the canine descending aorta endured the high-pressure condition without ruptures or aneurysmal changes.
In addition, PEG may provide more durability in future protocols. PEG is polymerized by gamma irradiation [23]. The degree of cross-linking depends on the molecular weight and concentration of the polymer and the radiation dose and rate [23]. With increasing polymerization by gamma rays, the viscosity of liquid PEG increases, it transforms into a gel, and finally becomes a solid. In our preliminary study, the PEG solution (80% PEG 1000 MW) was not gelatinized with 100 kGy gamma irradiation (data not shown). However, if an appropriate irradiation dose and PEG molecular weight for gelatinization can be found without compromising the decellularization, the PEG gel coating should increase the durability of the decellularized valve.
In terms of recellularization, the grafts were well repopulated by the host cells. At 2 months after implantation into the dogs, the surface of the graft was completely covered with a monolayer of ECs. In addition, several layers of vimentin-positive cells lay beneath the ECs. Since vimentin is positive in immature mesenchymal cells, we believe those cells might be precursor cells of smooth muscle cells. Indeed, abundant smooth muscle cells were seen at 6 months after implantation in the same area. The regeneration of the vascular wall was excellent, as demonstrated by the completely regenerated ECs and vasculogenesis seen in the tissue 6 months after implantation. This recellularization with site-specific cells indicates that our graft was a good scaffold for proper remodeling. The results of quantitative RT-PCR experiments supported these histologic findings.
There are several limitations to this study:
First, because the animals used in this study did not have anti-1.3-Gal antibodies, no evaluation of the hyperacute rejection induced by 1.3-Gal could be performed. To evaluate it accurately, we need to implant these valves into Old World monkeys, such as rhesus monkeys or baboons, that have -1.3 Gal antibodies [24].
Second, no hemodynamic functional evaluation of the grafts was performed. We plan to use an aortic root replacement model in juvenile sheep to evaluate the hemodynamic function and further verify remodeling of our grafts.
Third, although our grafts showed a potential to be a good scaffold for regeneration, the effect of gamma irradiation to cross-linking of collagen in the graft was not evaluated. Further analyses are needed.
Fourth, our mechanical strength results are tentative because they came not from a biaxial tensile test but from a uniaxial tensile test.
Fifth, no control study in the dog experiments was performed. The number of dogs at 6 months after implantation was small, which impairs the persuasiveness of the study.
In conclusion, we developed a new method of decellularization with PEG and gamma irradiation. The tissues treated with our decellularization process possessed low immunogenicity, were little subject to calcification, and demonstrated acceptable durability and recellularization. The DNA sequences of PERV the oligosaccharide chains of 1.3-Gal were eliminated through the decellularization process, which indicates that the decellularization process could reduce risk of unknown viral infections and acute rejection in xenotransplantation.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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-irradiation on methoxy-poly(ethylene glycol)s in aqueous solutions J Bioact Compat Polym 1998;13:279-302.[Abstract]This article has been cited by other articles:
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  T. Ota, T. W. Gilbert, D. Schwartzman, C. F. McTiernan, T. Kitajima, Y. Ito, Y. Sawa, S. F. Badylak, and M. A. Zenati A fusion protein of hepatocyte growth factor enhances reconstruction of myocardium in a cardiac patch derived from porcine urinary bladder matrix. J. Thorac. Cardiovasc. Surg., November 1, 2008; 136(5): 1309 - 1317. [Abstract] [Full Text] [PDF]
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 P. Akhyari, H. Kamiya, A. Haverich, M. Karck, and A. Lichtenberg Myocardial tissue engineering: the extracellular matrix. Eur. J. Cardiothorac. Surg., August 1, 2008; 34(2): 229 - 241. [Abstract] [Full Text] [PDF]
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 P. M. Dohmen and W. Konertz Decellularization of xenogenic biologic tissue. Ann. Thorac. Surg., June 1, 2008; 85(6): 2163 - 2163. [Full Text] [PDF]
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T. Ota, Y. Okita, and Y. Sawa Reply. Ann. Thorac. Surg., June 1, 2008; 85(6): 2163 - 2164. [Full Text] [PDF]
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) show the margin between the vascular walls and the subcutaneous tissue. (A) The invasion of inflammatory cells was not seen in the decellularized tissue, (B) but invading inflammatory cells (}) were seen in the porcine native tissue (hematoxylin and eosin stain, original magnification x100). Calcification analysis is shown in C and D. (C) A small amount of calcification was observed in the decellularized tissue, but (D) much stronger calcification was seen in the glutaraldehyde-treated porcine tissue (von Kossa stain, original magnification x40).