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J Thorac Cardiovasc Surg 2003;126:498-503
© 2003 The American Association for Thoracic Surgery
Surgery for congenital heart disease |
a Department of Cardiothoracic Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, Calif, USA
Received for publication May 29, 2002; revisions received June 18, 2002; revisions received October 25, 2002; accepted for publication December 9, 2002.
* Address for reprints: Winfield J. Wells, MD, Department of Cardiothoracic Surgery, Childrens Hospital Los Angeles, 4650 Sunset Blvd, MS #66, Los Angeles, CA 90027, USA
wwells{at}chla.usc.edu
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Abstract |
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METHODS: The superior vena cava was replaced in 8 minipigs with either a polytetrafluoroethylene interposition graft (2 pigs) or a depopulated (acellular), cryopreserved superior vena caval homograft (6 pigs). After 6 months, the animals were killed, and the grafts were examined for patency and histology, including immunostaining. No anticoagulation was used.
RESULTS: Polytetrafluoroethylene grafts have a cross-sectional luminal narrowing, ranging from 16% to 40%. Histology showed only partial intimal ingrowth, with excessive subendothelial fibrosis and early calcification. In contrast, the depopulated venous homografts showed minimal luminal narrowing, ranging from 2% to 9%. These grafts were completely repopulated by the recipient with an endothelial lining, which stained positively for factor VIII, and a subendothelial region appropriately recellularized by myofibroblasts, which stained positively for smooth muscle actin and procollagen. There was no evidence of an immune response to the venous homografts, as judged by staining for T-cell surface antigen, CD4, and CD8. Thrombus was not seen in any of the grafts.
CONCLUSION: Depopulated, cryopreserved vena caval homografts might be superior conduits for cavopulmonary connection during completion of the Fontan operation by using the extracardiac conduit technique.
Until recently, the need for replacement of large veins was limited and usually involved tumor resection or trauma. The recent advent of the extracardiac Fontan procedure with inferior cavopulmonary connection requiring a conduit extension has raised interest in large-caliber venous vascular prostheses.
Grafting of a larger-caliber vein has usually been performed with a polytetrafluoroethylene (PTFE) graft with or without external ring support. Although the results have been satisfactory, complete re-endothelialization of these conduits has been questionable. Without a well-healed endothelium, such conduits are at risk for platelet fibrin aggregates that lead to fibrointimal thickening, as well as possible thromboembolism. For this reason, anticoagulation, with its attendant risks, has often been used after PTFE grafting.
Cryopreserved homograft tissue is an alternate choice for major vein replacement. Because of the diameter and length of the graft that is usually needed, aortic tissue has been used most frequently. Unfortunately, aortic homografts have tended to degenerate over time and develop a thickened and calcified vessel wall that could become obstructive or thrombogenic. Several studies have suggested that this process might be due to the homograft recipient’s immune response to the viable homograft donor cells.1-3
Antigen reduction of homograft tissue by means of cell-depopulation techniques has recently been introduced. Depopulated pulmonary homografts have been shown to be less antigenic than their viable counterparts.4 In animal studies these depopulated conduits have shown rapid repopulation with functioning host cells and a significantly decreased tendency to degenerate and calcify.5-7 A large-diameter venous conduit that would repopulate with recipient cells to form a normally functioning vascular endothelium and a renewable connective tissue matrix would be ideal. We have therefore investigated depopulated cryopreserved vena caval homograft tissue used to replace the superior vena cava (SVC) in a large-animal model.
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Materials and methods |
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Preparation of depopulated SVC allografts
The SVC was harvested from 6 juvenile farm pigs (diameter, 18-20 mm) and then transported in iced physiologic buffer for depopulation processing and cryopreservation. The tissue was treated by using a propriety method (SynerGraft; CryoLife, Inc, Kennesaw, Ga) to eliminate cells in the vessel wall. The steps included cell lysis in hypotonic solution, enzymatic digestion of nucleic acid, and washout in an isotonic neutral buffer. Once depopulated, the veins were cryopreserved and stored in liquid nitrogen until implantation.
SVC replacement
SVC replacement was carried out through a modified manubrium-sparing sternotomy. After heparinization, vascular clamps were secured, and the SVC was resected. The native SVC was between 16 and 18 mm in diameter. The SVC was then replaced, with the anastomosis completed with a 6-0 running polypropylene suture technique.
Two pigs received an 18-mm PTFE graft 3 to 4 cm in length. Six pigs had SVC replacement with a depopulated cryopreserved SVC homograft that was 18 to 20 mm in diameter and 3 to 4 cm in length. The animals were given a 5-day course of prophylactic antibodies postoperatively (first-generation cephalosporin).
All of the animals survived the operation and were maintained without anticoagulation. One animal died from pneumonia at an interval of 2 months after the operation. Unfortunately, the SVC was not explanted in a timely manner and was lost for analysis. The remaining 7 animals survived to 6 months, at which time they were killed, followed by removal of their SVC grafts by means of re-entry sternotomy.
Microscopic analysis (histology-immunochemistry)
Specimens of porcine SVC were evaluated before and after depopulation treatment (SynerGraft) for histology and immunochemistry analysis.
The explanted SVC replacement grafts were dissected and divided between buffered neutral formalin or cryosectioning medium. The fixed specimens were then cut into proximal, mid, and distal conduit segments and then embedded in paraffin and stained with hematoxylin and eosin. Representative sections were examined for patency, with measurement of cross-sectional luminal narrowing assessed by using a planimetry technique.
A separate portion of the explanted graft wall was equilibrated in 15% sucrose in phosphate-buffered saline for at least 1 hour at 4°C and then frozen in embedding compound (O.C.T. compound; Tissue-Tek, Torrance, Calif) for cryosectioning and immunostaining. Monoclonal antibodies were used to make several determinations, including the following: for cellular immune response to the graft, T-cell surface antigen (anti-TCR 1 86D; VMRD, Pullman, Wash), cytotoxic T cells (anti-CD8; Zymed Labs, San Francisco, Calif), and helper T cells, macrophages, and other inflammatory cells (anti-CD4; Center for Animal Biotechnology, University of Melbourne, Melbourne, Australia); for cellular identification and function, smooth muscle actin (IgG2A; Vector, Burlingame, Calif), endothelial cells (anti-factor VIII; Sigma, St Louis, Mo), monocytes and macrophages (DH59B, VMRD, Inc), class I and class II major histocompatibility antigens (anti-MHC I and II, VMRD, Inc), and procollagen type I production (anti-procollagen SP1.D8; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa).
Antibody binding was visualized colorimetrically by using peroxidase-conjugated antibody and reaction with 3, 3-diaminobenzimide.
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Results |
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Microscopic appearance of normal and depopulated vena caval tissue
Hematoxylin and eosin–stained sections of the normal and depopulated cryopreserved porcine SVC tissues were obtained (Figure 2).
Untreated tissue showed a normal cellular content and distribution pattern. The depopulation treatment resulted in a near-total removal of histologically demonstrable cells throughout the tissues. What remained was connective tissue matrix, which was primarily composed of structural collagen, elastin, and smooth muscle actin.
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Immunostaining of preimplantation depopulated cryopreserved SVC showed virtually no reaction to any of the antibodies used.
Assessment of luminal narrowing of postimplantation SVC grafts
Cross-sectional luminal area was measured within the proximal, mid, and distal regions of the SVC replacement grafts when explanted at 6 months. Maximal narrowing was noted to be 16% and 40% for the 2 PTFE control animals. The maximal cross-sectional luminal narrowing for the depopulated cryopreserved SVC allograft was a median of 6% and ranged from 2% to 9% among the 5 pigs that survived to 6 months.
Microscopic appearance of explanted SVC grafts
The 2 PTFE grafts showed only partial intimal ingrowth, with excessive fibrosis and early calcification. There was a paucity of endothelial cells, which formed an inconsistent lining. The thickened ingrowth stained positively for smooth muscle actin.
Histology determined by means of hematoxylin and eosin staining of the postimplantation depopulated cryopreserved SVC allografts demonstrated re-establishment of a normal-appearing cellular pattern with microvasculature throughout. The appearance was comparable with that of the normal porcine SVC tissue prepared as a control. Evaluation with immunohistochemical stains demonstrated factor VIII–positive cells (endothelial cells), smooth muscle actin, MHC I and MHC II, and granulocytes and macrophages (Figure 3). The explanted grafts showed the same pattern of staining as seen in the normal SVC. Endothelial cells were demonstrated in the lumen around the entire graft and around microvascular structures. Smooth muscle actin–positive cells were found throughout the intimal lining, arterioles, and venules in the appropriate pattern.
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Cellular biosynthetic function was demonstrated with procollagen type I staining. The normal SVC control had a very low level of procollagen production, which was found primarily in a small number of cells scattered in the adventitia. The explanted homografts showed enhanced procollagen production, with some areas of numerous positively stained cells located primarily in the graft media.
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Discussion |
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Several studies have documented the outcomes of PTFE used for prosthetic replacement of the thoracic vena cava. Animal studies by Fiore and colleagues10 reported in 1982 showed excellent short-term patency of PTFE SVC grafts and suggested that grafts with external ring support might be superior. The potential advantage of spiral supported grafts was further demonstrated by Masuda and coworkers11 in long-term animal studies. These showed better patency and more favorable re-endothelialization at 3 years among spiral supported versus unsupported PTFE conduits.
Investigation into the healing process of PTFE grafts used for replacement of large veins has been studied in animals by Kogel and colleagues.12 Primary formation of intramural thrombogenic matrix in the prosthetic wall was thought to induce tissue and capillary invasion from surrounding tissue by means of chemotactic release of growth factors. This in turn led to multifocal endothelial cell ingrowth from transprosthetic channels.
Dartvelle and associates13 have reported on the long-term performance after PTFE graft replacement of the SVC in patients undergoing resection of mediastinal tumors. Among 15 patients followed for a median of 31 months, the 3- and 5-year actuarial graft patency was 86%. Of interest is the authors’ recommendation that patients be given warfarin sodium (Coumadin) for 6 months after graft placement.
Marcelletti and coworkers8 have provided long-term follow-up of PTFE conduits used for extracardiac Fontan repair. Serial magnetic resonance imaging studies carried out in 30 patients showed a mean reduction of the internal conduit diameter of 18% ± 8% during the first 6 months of implantation. There was no progression over the following 5 years. Even the maximal internal diameter reduction of 32% ± 8% found among the 10 patients with the most obstruction did not progress over time. It is of interest that the 2 PTFE conduits placed in our pigs had luminal narrowing of 16% and 40%, respectively, which falls within the range of what has been observed clinically by Marcelletti and coworkers.
Although there are a few reports9 of the use of cryopreserved aortic homograft tissue for vena caval replacement, there is no reported long-term follow-up on the fate of this conduit. In general, aortic homograft tissue has shown a tendency to degenerate, calcify, and become obstructive when used in the right ventricular outflow tract. Structural degeneration is also known to occur more frequently and earlier in younger recipients.14,15 Evidence is mounting that cryopreserved homograft deterioration might be due to both a cellular and humoral immune response of the recipient.16 However, there is no agreement on the relationship of the immune response as measured by antibody production on graft function, with some authors3 demonstrating a correlation between antigen production and structural degeneration and others17,18 finding no correlation. What does seem to be clear is that standard cryopreserved homografts tend to lose donor cellularity and do not revascularize or recellularize from the recipient.19 Thus these conduits would not be expected to develop the antithrombogenic effects of re-endothelialization nor would they regenerate their connective tissue matrix.
In contrast to standard cryopreserved homograft tissue, depopulated conduits show virtually no humoral immune response in patients, as measured by means of Panel-reactive antibody analysis, at 1 and 3 months after implantation.4 By using an animal model, a process of recellularization and adaptive remodeling has also been shown to take place in these depopulated grafts. This was true both for depopulated allograft and xenograft valved conduits, in which functional repopulation of the matrix, including leaflet tissue, was demonstrated.20
It is our belief that a conduit that has been repopulated with functional recipient cells will be the optimal graft for replacing or extending major veins. Whether these grafts will have the ability to grow remains in question and is the subject of ongoing study. The need for anticoagulation might also be mitigated by the adaptive remodeling of an intact endothelium, although this remains to be proved. Whether anticoagulation should be managed with an antiplatelet agent or with warfarin has not yet been resolved. Anticoagulation would most likely be suggested for several months while repopulation was in progress. Avoidance of anticoagulation is of particular importance in children, in whom achieving a consistently increased clotting time is often difficult.
On the basis of our experience in this animal study, we believe that depopulated cryopreserved homograft tissue is the best alternative for major vein grafting. Clinical use of this technique is under consideration.
Because the number of animals available for investigation was limited, it was elected to do only 2 control studies with PTFE conduits. This number was believed to be adequate because, as noted above, a number of animal studies have evaluated PTFE grafts in similar SVC replacement models. Additionally, human studies have documented the outcomes of PTFE venous grafts, and the results in our 2 control animals were similar to what has been previously found. Thus, we concluded that our model was valid for demonstrating problems with graft obstruction if there was a propensity for this to occur. It must be acknowledged, however, that the number of PTFE control animals was very small and could not yield statistically significant data.
Conduits were in place for 6 months before explantation and investigation. It is possible that there could be degeneration and progressive obstruction over longer time periods; however, as noted in our discussion, the literature suggests that narrowing of major vein conduits appears to occur in the first 6 months after implantation and does not significantly progress between that point and 3 years. It was this information that led us to investigate our grafts at 6 months. It is possible, however, that with longer follow-up, additional obstruction, thrombus, or both could form. Experience with standard cryopreserved homografts suggests that destruction and obstruction is an ongoing event. Therefore, longer-term follow-up is needed.
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Discussion |
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If I could set the basis for doing this study, have you, in your clinical series, had issues with clotted PTFE grafts or early failure with your grafts? Do you use aspirin or warfarin early on in your clinical practice, and did you do so with these piglets? I guess most people would use aspirin, if not warfarin, at least early in the postoperative course.
What was the size mismatch relative to the adult human? For example, we have tried to put in at least a 22-mm graft as an extracardiac conduit. Was this a size mismatch that might have created flow-and-eddy problems that could potentially create early stenosis issues? I am wondering about the size mismatch being a cause. Setting that aside, potentially where will you harvest from the human if the organs that are taken are the liver and the heart? What portion of the cava is available, the renal, the suprarenal?
Dr Wells. Thank you for the questions.
We have not really observed a problem with thrombosis. We were concerned about the reports coming from Italy that there might be up to a 30% to 40% cross-sectional luminal reduction in the serially magnetic resonance imaging–;studied vena caval extensions for Fontan completion. Therefore that in and of itself was the impetus to determine whether there is something better. Also, we have been concerned in our practice about leaving patients without aggressive anticoagulation for the first at least 6 months after putting in the PTFE graft. Therefore, you are going to subject a child to at least 6 months of anticoagulation, which you might be able to avoid. And I should add that in these studies there was no anticoagulation. Thanks for pointing that out.
As far as the size of the homografts, we tried to use clinically appropriate sizes. Therefore, these were 18- to 20-mm homografts, 3 cm to 4 cm in length, and we think that the typical patient in whom we are doing this for Fontan completion gets a conduit of 18 to 20 mm and about 5 cm in length. Therefore we believed that this was clinically appropriate.
Finally, either the innominate vein in a larger donor or potentially the inferior vena cava, which will have more branches, could be sites. The infrarenal cava could be a site for harvesting, and we are just getting interested in investigating the best place from which to take that venous tissue.
Dr Vaughn A. Starnes (Los Angeles, Calif). You are implying, then, that this would be homograft tissue and not xenograft tissue?
Dr Wells. There is evidence currently that there is no difference between homograft and xenograft for the depopulated tissues. That is animal work that is fairly convincing. However, I think if you want to use this clinically during our foreseeable lifetime, we probably need allografts. We probably need human tissue to get this going.
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Footnotes |
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References |
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This article has been cited by other articles:
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  K. J. Zehr, M. Yagubyan, H. M. Connolly, S. M. Nelson, and H. V. Schaff Aortic root replacement with a novel decellularized cryopreserved aortic homograft: Postoperative immunoreactivity and early results J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1010 - 1015. [Abstract] [Full Text] [PDF]
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