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Ann Thorac Surg 2001;71:S433-S436
© 2001 The Society of Thoracic Surgeons

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Basic research

Transformation of nonvascular acellular tissue matrices into durable vascular conduits

^a CryoLife Incorporated, Kennesaw, Georgia, USA
^b Cardiovascular Research Laboratory, East Carolina University, Greenville, North Carolina, USA

Address reprint requests to Dr Ollerenshaw, CryoLife Inc, 1655 Roberts Blvd NW, Kennesaw, GA 30144
e-mail: ollerenshaw.jeremy{at}cryolife.com

Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.

	Abstract

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Prosthetic grafts commonly used for vascular reconstruction are limited to synthetics and cross-linked tissue grafts. Within these devices, graft infections are common, compliance mismatch is significant, and handling qualities are poor. Natural biological tissues that are unfixed have been shown to resist infections and be durable and compliant. A natural biological matrix that could be remodeled appropriately after implantation would be a desirable graft for vascular reconstruction.

Methods. SynerGraft tissue engineering strategies have been used to minimize antigenicity and produce stable unfixed vascular grafts from nonvascular bovine tissues. These grafts have replaced the abdominal aortas of 8 dogs that have been followed for up to 10 months.

Results. Early evaluation indicates rapid recellularization by recipient smooth muscle actin positive cells, which become arranged circumferentially, into the media. Arterioles were present in the adventitial areas and endothelial cells were seen to cover lumenal surfaces. After 10 months, grafts were patent and not aneurysmal.

Conclusions. These data indicate that SynerGraft processing of animal tissues is capable of producing stable vascular conduits that exhibit long-term functionality in other species.

	Introduction

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Currently, clinical repair of the aorta and peripheral vessels or construction of arteriovenous shunts is usually performed using synthetic graft materials. These materials, such as Dacron or polytetrafluoroethylene, have poor compliance and low patency rates when small diameters are used and are more easily colonized by bacteria than biological tissues. Additionally, the handling characteristics of these grafts are poor. Alternative graft materials are clearly needed.

Previously, animal tissues were used as vascular grafts in an attempt to avoid the disadvantages of synthetic materials. Bovine carotid arteries, internal mammary arteries [1–7], and ovine collagen tubular matrices grown in vivo [8, 9] have all been used clinically as vascular conduits for hemodialysis access and coronary or peripheral arterial bypass. These grafts have a close compliance match to native blood vessels and therefore exhibit better handling characteristics. Bovine carotid [1–6] and internal mammary arteries [2, 7] have been used in humans. These xenografts were chemically treated with glutaraldehyde to cross-link the tissue protein and reduce or mask antigenicity. Treating tissues in this way makes the grafts nonviable and unable to be remodeled by the host. Performance of many glutaraldehyde-treated vascular grafts has been disappointing. Common modes of failure include aneurysm formation because of weaknesses in the conduit wall [1], and conduit calcification leading to loss of hemodynamic compliance and stenosis [1]. An external, reinforcing mesh has been added in some cross-linked bovine artery grafts that has led to a reduction in aneurysm formation, but patency issues make these grafts less than ideal.

An ovine graft of nonvascular origin has also been used clinically [8, 9]. This cross-linked collagen graft fabricated from the scarring around a small diameter mandril has been used for hemodialysis access and peripheral arterial bypass. As with other chemically fixed animal tissues, structural deterioration of the graft wall with dilation and aneurysm formation is a problem. This deterioration might indicate that cross-linking is unable to reduce the recognition of xeno-antigens to an acceptable level and further supports the assertion that the use of cross-linking agents inhibits the adoption of the natural tissue matrix as a structural scaffold by the host.

To produce a structurally viable biological matrix for use as a vascular graft, antigen reduction with preservation of the matrix for remodeling is necessary. Accordingly, we have used several methods to render bovine tissues nonimmunogenic before implantation. The principal method is called SynerGraft processing (patented by CryoLife Inc, Kennesaw, GA) [10, 11]. SynerGraft methods focus on native cell removal from the collagen tissue matrix. Removal of cell material theoretically will reduce or eliminate the immunologic response mounted by the recipient and leave a functional vascular matrix that is available for autogenous remodeling. Migration of the recipient’s tissue specific cells into the matrix might eventually render the graft indistinguishable from other endogenous tissues.

Conduits developed using the described tissue engineering technologies are suitable for many applications involving vascular grafts, therefore precluding the need for traumatic harvest of autologous tissues. Because the grafts are biological, they handle like native tissues. Additionally, remodeling processes might result in an active blood supply to the new tissue and therefore lead to resistance to bacterial colonization.

Using bovine tissues, we have produced tissue-engineered matrix conduits that have been implanted into the infrarenal aorta of dogs. Studies designed to evaluate durability, patency, and histologic fate were performed during a 10-month follow-up period.

	Material and methods

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Bovine ureters were used to provide the conduit matrix for the tissue-engineered vascular graft. These tissues are available in lengths and diameters suitable for a number of vascular applications and they do not contain valves in the lumen or possess tributaries that would require ligation. Ureters were obtained from United States Department of Agriculture-approved slaughterhouses. The tissues were washed in physiologic salt solution and transported for tissue preparation on ice within 24 hours of harvest. Ureters were first dissected free of adherent connective tissue and fat and only segments with a 6-mm internal diameter were used for further processing.

Initial processing consisted of bioburden reduction using a solution of multiple antibiotics. Removal of more than 95% of all cellular material from the ureter matrix was thus achieved in several steps. First, incubation in sterile water produced hypotonic cell lysis. The tissue was then equilibrated in buffer and treated with a solution containing ribonuclease and deoxyribonuclease. An isotonic washout over several days completed the cellular protein removal. Removal of cellular debris was monitored using hematoxylin and eosin staining of histologic sections. Tissues were then sterilized by gamma irradiation before use and analysis of sterility was carried out on each processing batch.

Eight mongrel dogs weighing 50 to 60 lb were anesthetized with sodium thiopental, endotracheally intubated, and placed on inhaled isoflurane. The abdomens were prepared and draped in sterile fashion. A midline incision was made and the abdominal aorta distal to the renal arteries was isolated in each dog. SynerGraft vascular conduits were prepared by washing the tissue in 100 mL of sterile HEPES-buffered Dulbecco’s modified Eagle medium and a segment approximately 6 cm in length and 6 mm in internal diameter was inserted as an aortic interposition graft using interrupted Prolene (Ethicon, Somerville, NJ) sutures to construct proximal and distal end-to-end anastomoses. All animals received 325 mg aspirin and 75 mg dipyridamole orally daily for 2 days before and 14 days after the operation.

Patency and structural stability were observed with angiographic examination after the operation every 6 weeks in the longer survivors and once immediately before euthanasia. Two animals were sacrificed at 3 weeks, 3 at 6 weeks, and 1 at 13 weeks after the operation. The 2 remaining animals were last evaluated at 43 weeks and are still living. After sacrifice, grafts were removed in bloc incorporating proximal and distal anastomoses, inspected grossly and processed for histologic analysis.

After harvest, grafts were perfusion fixed at 100 mm Hg pressure in 10% buffered formaldehyde solution. The whole of the graft along with anastomotic sites and proximal and distal native aorta was divided into seven tissue segments and placed in paraffin blocks for processing. Hematoxylin and eosin-stained sections of these tissues were examined and immunohistochemical analysis was carried out using specific antibodies (Zymed Laboratories, Inc, South San Francisco, CA) to identify the presence of alpha-smooth muscle actin (alpha-SMA), desmin, and vimentin contractile filaments.

All animals in this study received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).

	Results

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
After processing, tissue-engineered vascular grafts from bovine ureter showed removal of more than 95% of bovine cellular material. The remainder consisted of cellular debris and not intact cells (Fig 1). Conduit graft sterility and pyrogen levels of less than 20 endotoxin units were demonstrated. Implantation of these interposition grafts into the canine infrarenal aorta was uncomplicated and handling properties of the grafts were similar to normal vascular tissue.

View larger version (79K): [in this window] [in a new window]   Fig 1. Sections of bovine ureter (a) before and (b) after SynerGraft processing to remove bovine tissue antigen. Smooth muscle, fibroblasts, and epithelial cells are removed. (Hematoxylin and eosin; original magnification x100.)

View larger version (176K): [in this window] [in a new window]   Fig 2. Tissue sections showing revitalization of acellular bovine vascular conduit after 13 weeks of implantation. Sections show the circumferential appearance of cells present in the graft media and indicate endothelial cells present on the lumenal surface. (Hematoxylin and eosin; original magnification x400.)

Immunohistochemistry staining was used to identify the type of cells present in the recellularized grafts. The proportion of cells expressing smooth muscle contractile proteins were demonstrated using stains containing antibodies to alpha-SMA, desmin, and vimentin. A large percentage of medial cells at 3, 6, and 13 weeks, were alpha-SMA positive. Vimentin was also commonly expressed by alpha-SMA-positive cells. Desmin-positive cells were less abundant but present in a subpopulation. Most of the cells present in the grafts stained positive for at least one of these contractile proteins. As no intact cells were present in the SynerGraft conduits before implant, all cell-specific immunostaining demonstrable for alpha-SMA, desmin, and vimentin was present on cells that had originated from the host.

	Comment

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
An ideal vascular graft material is not readily available. Using SynerGraft technology, a natural biological conduit matrix that has handling properties similar to natural tissue and that might be optimal for a variety of vascular applications has been developed. Our tissue-engineered graft material is carefully chosen animal conduit from which we are able to create a biological tissue matrix by a process of carefully orchestrated xeno-antigen removal. The matrix then acts as a scaffold for spontaneous repopulation by host cells in vivo, leading to tissue reconstruction and stabilization. The result is a fully functional, nonimmunogenic, viable vascular conduit containing autologous cells expressing contractile proteins. The use of these conduits as aortic grafts in dogs demonstrates for the first time, fully functional tissue-engineered vascular grafts over a prolonged period of 10 months.

There have been animal tissues used as vascular grafts previously, but all have been cross-linked before use to preserve the tissue and mask xeno-antigens. These grafts have not performed well. However, removal of xeno-antigen as an engineering approach has proved successful in this study. Also, recent work has shown SynerGraft technology to enable the successful grafting of porcine heart valves into sheep [12] and clinically into humans [13] without the use of immunosuppressive agents. In all of the SynerGraft implant scenarios, xenogenic tissues were spontaneously recellularized by the host and resulted in functional grafts that contained distinct and organized populations of cells. Apparent from studying SynerGraft vascular tissues after explant is that the process of incorporation by the host is associated with capillary ingrowth. In the case of vascular grafts, this might produce resistance to infection as is seen in other biological grafts. As opposed to current synthetic graft materials, this biological graft may be used as a primary graft or in a contaminated field with the potential for reduced risk of infection. When used for hemodialysis access, the ability of a graft to seal rapidly after needle puncture is desirable. Biological grafts such as the SynerGraft conduit could be expected to become hemostatic more quickly than synthetic materials. Finally, resistance to graft kinking, an additional property of this material, gives the SynerGraft conduit an advantage in peripheral bypass where crossing a joint is involved.

SynerGraft vascular conduits have potential advantages over existing synthetic and cross-linked animal tissue grafts in clinical situations involving vascular access or peripheral, coronary, and other vascular replacement or bypass. These tissue-engineered grafts may be used as primary grafts and eliminate the need for traumatic autologous tissue harvest for vascular procedures.

	Acknowledgments

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
CryoLife, Inc, provided financial support for the study. The authors are grateful for the excellent technical assistance of Christy L. Goodman and Cindy K. Davenport.

	Footnotes

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Drs Clarke, Black, and Ollerenshaw have a financial relationship with CryoLife, Inc.

	References

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 

1. Schmitz-Rixen T., Megerman J.M., Warnock D.F., et al. Long-term study of a compliant biological vascular graft. Eur J Vasc Surg 1991;5:149-158.[Medline]
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3. Schroder A., Imig H., Peiper U., et al. Results of a bovine collagen vascular graft (Solcograft-P) in infra-inguinal positions. Eur J Vasc Surg 1988;2:315-321.[Medline]
4. Guidoin R., Domurado D., Couture J., et al. Chemically processed bovine heterografts of the second generation as arterial substitutes: a comparative evaluation of three commercial prostheses. J Cardiovascular Surg 1989;30:202-209.[Medline]
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9. Koch G., Gutsche S., Pascher O., Fruhwirth H., Glanzer H. Analysis of 274 Omniflow vascular prostheses implanted over an eight-year period. Aust N Z J Surg 1997;67:637-639.[Medline]
10. Goldstein S, inventor; CryoLife Inc, assignee. Method of preparing transplant tissue to reduce immunogenicity upon implantation. US patent 5,613,982. 1997.
11. Goldstein S, inventor; CryoLife Inc, assignee. Treated tissue for implantation and methods of preparation. US patent 5,632,778. 1997.
12. O’Brien M.F., Goldstein S., Walsh S., Black K.S., Elkins R., Clarke D. The SynerGraft valve: a new Acellular (nonglutaraldehyde-fixed) tissue heart valve for autologous recellularization first experimental studies before clinical implantation. Semin Thorac Cardiovasc Surg 1999;11:194-200.[Medline]
13. Goldstein S., Clarke D.R., Walsh S.P., Black K.S., O’Brien M.F. Transpecies heart valve transplant: advanced studies of a bioengineered xeno-autograft. Ann Thorac Surg 2000;70:1962-1969.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Clarke, D. R. Articles by Ollerenshaw, J. D. Search for Related Content PubMed PubMed Citation Articles by Clarke, D. R. Articles by Ollerenshaw, J. D. Related Collections Cardiac - pharmacology Peripheral vascular